de Duve was born of a shopkeeper Alphonse de Duve and wife Madeleine Pungs in the village of Thames Ditton, near London. His parents fled Belgium at the outbreak of the First World War. After the war in 1920, at age three, he and his family returned to Belgium. He was a precocious boy, always the best student (primus perpetuus as he recalled) in school, except for one year when he was pronounced “out of competition” to give chance to other students. He was educated by the Jesuits at Onze-Lieve-Vrouwinstituut in Antwerp, before studying at the Catholic University of Leuven in 1934. He wanted to specialize in endocrinology and joined the laboratory of the Belgian physiologist Joseph P. Bouckaert. During his last year at medical school in 1940, the Germans invaded Belgium. He was drafted to the Belgian army, and posted in southern France as medical officer. There, he was almost immediately taken as prisoner of war by Germans. But fortunate of his ability to speak fluent German and Flemish, he outwitted his captors and escaped back to Belgium. (The adventure he later described as “more comical than heroic”.) He immediately continued his medical course, and obtained his MD in 1941 from Leuven. His primary research was on insulin and its role in glucose metabolism. He made an initial discovery that a commercial preparation of insulin was contaminated with another pancreatic hormone, the insulin antagonist glucagon. However, laboratory supplies at Leuven were in shortage, he therefore enrolled in a programme to earn a degree in chemistry at the Cancer Institute. His research on insulin was summed up in a 400-page book titled Glucose, Insuline et Diabète (Glucose, Insulin and Diabetes) published in 1945, simultaneously in Brussels and Paris. The book was condensed into a technical dissertation which earned him the most advanced degree at the university level agrégation de l’enseignement supérieur (an equivalent of a doctorate – he called it “a sort of glorified Ph.D.”) in 1945. His thesis was followed by a number of scientific publications. He subsequently obtained MSc in chemistry in 1946, for which he worked on the purification of penicillin. To enhance his skill in biochemistry, he trained in the laboratory of Hugo Theorell (who later won The Nobel Prize in Physiology or Medicine in 1955) at the Nobel Medical Institute in Stockholm for 18 months during 1946-1947. In 1947 he received a financial assistance as Rockefeller Foundation fellow and worked for six months with Carl and Gerti Cori’s at Washington University in St. Louis (the husband and wife were joint winners of The Nobel Prize in Physiology or Medicine in 1947).

In March 1947 de Duve joined the faculty of the medical school of the Catholic University of Leuven teaching physiological chemistry. In 1951 he became full professor. In 1960 Detlev Bronk, the then president of the Rockfeller Institute (what is now Rockefeller University) of New York City, met him at Brussels and offered him professorship and a laboratory. The rector of Leuven, afraid of entirely losing de Duve, made a compromise over dinner that de Duve would still be under part-time appointment with a relief from teaching and conducting examinations. The rector and Bronk made an agreement which would intilally last for five years. The official implementation was in 1962, and de Duve simultaneously headed the research laboratories at Leuven and at Rockefeller University, dividing his time between New York and Leuven. In 1969 the Leuven university was split into two separate universities. He joined the French-speaking side of Université catholique de Louvain. He took emeritus status at Université catholique de Louvain in 1985 and at Rockefeller in 1988, though he continued to conduct research. Among other subjects, he studied the distribution of enzymes in rat liver cells using rate-zonal centrifugation. His work on cell fractionation provided an insight into the function of cell structures. He specialized in subcellular biochemistry and cell biology and discovered peroxisomes and lysosomes, cell organelles.

His work has contributed to the emerging consensus that the endosymbiotic theory is correct; this idea proposes that mitochondria, chloroplasts, and perhaps other organelles of eukaryotic cells originated as prokaryote endosymbionts, which came to live inside eukaryotic cells.  de Duve proposed that peroxisomes may have been the first endosymbionts, which allowed cells to withstand the growing amounts of free molecular oxygen in the Earth’s atmosphere. Since peroxisomes have no DNA of their own, this proposal has much less evidence than the similar claims for mitochondria and chloroplasts. His later years were mostly devoted to origin of life studies, which he admitted as still a speculative field.

de Duve was brought up as a Roman Catholic. However his later years indicated inclination towards agnosticism, if not strict atheism. He was opposed to the notion of a creator. “It would be an exaggeration to say I’m not afraid of death,” he explicitly said to a Belgian newspaper Le Soir just a month before his death, “but I’m not afraid of what comes after, because I’m not a believer.” He strongly supported biological evolution as a fact, and dismissive of creation science and intelligent design, as explicitly stated in his last book, Genetics of Original Sin: The Impact of Natural Selection on the Future of Humanity. He was among the seventy-eight Nobel laureates in science to endorse the effort to repeal Louisiana Science Education Act of 2008.  de Duve married Janine Herman on 30 September 1943. Together they had had two sons, Thierry and Alain, and two daughters, Anne and Françoise. Janine died in 2008, aged 86.

de Duve won Francqui Prize for Biological and Medical Sciences in 1960, and Nobel Prize for Physiology or Medicine in 1974. King Baudouin of Belgium honoured him to Viscount in 1989. He was the recipient of the Canada Gairdner International Award in 1967, and the Dr. H. P. Heineken Prize in 1973 from the Royal Netherlands Academy of Arts and Sciences. He was elected a foreign associate of the US National Academy of Sciences in 1975, and won the E. B. Wilson Award from the American Society for Cell Biology in 1989. He was also a member of the Royal Academies of Medicine and the Royal Academy of Sciences, Arts, and Literature of Belgium; the Pontifical Academy of Sciences of the Vatican; the American Academy of Arts and Sciences; the Academy of Sciences of Paris; the Deutsche Akademie der Naturforscher Leopoldina; and the Royal Society of London. In addition, he received honorary doctorates from eighteen universities around the world.

de Duve died on 4 May 2013, at his home in Nethen, Belgium, at the age of 95. He decided to end his life by legal euthanasia, performed by two doctors before his four children. He had been long suffering from cancer and atrial fibrillation, and his health problems were exacerbated by a recent fall in his home. He is survived by two sons and two daughters; two brothers, Pierre and Daniel; seven grandchildren; and two great-grandchildren.