John Eleuthère du Pont was born on November 22, 1938, in Philadelphia, Pennsylvania. He was the youngest of four children in his family. His parents were William du Pont, Jr. and Jean Liseter Austin. John grew up at Liseter Hall, a large mansion in Newtown Square, Pennsylvania. The mansion was built by his maternal grandfather in 1922 and sat on over 200 acres of land given to his parents as a wedding gift.

John's parents had successful families that emigrated from Europe to the United States in the early 19th century. In the 1920s and 1930s, both of his parents worked to acquire more land and develop Liseter Hall Farm. This farm was known for breeding and racing Thoroughbred horses. In 1941, when John was just two years old, his parents divorced. After the divorce, John's mother continued to run the farm and added a dairy herd of Guernseys and bred Welsh ponies.

John had two older sisters named Jean and Evelyn, and an older brother named Henry E. I. du Pont. He also had a younger half-brother named William du Pont III from his father's second marriage. John attended Haverford School and graduated in 1957. He then enrolled at the University of Pennsylvania and joined the Zeta Psi fraternity. However, he left the university before completing his first year.

Later, he attended college in Miami, Florida. There, he studied zoology under scientist Oscar T. Owre. He graduated from the University of Miami in 1965 with a Bachelor of Science degree. John continued his education and earned a doctorate in natural science from Villanova University in 1973.

While studying for his doctorate, John du Pont went on scientific expeditions to the Philippines and the South Pacific. He became known as an ornithologist, studying birds, and he is credited with discovering two dozen bird species. In 1957, he founded the Delaware Museum of Natural History and served on its board for many years. The museum officially opened to the public in 1972, and John played a role in its development.

In his personal life, John du Pont married Gale Wenk, an occupational therapist, on September 3, 1983. They met after he injured his hand in a car accident. However, their marriage lasted less than six months, and he filed for divorce ten months later.

John had various interests, including philately, which is the collection of stamps. He was known for buying rare stamps, including one that he purchased for $935,000 in 1980. This stamp later sold for over $9 million after his death. He also developed Liseter Hall Farm into a training facility for wrestling, calling it "Foxcatcher Farm" after his father's racing stable. Here, he sponsored amateur athletes and established a wrestling team.

On January 26, 1996, John du Pont shot and killed Olympic wrestler Dave Schultz in the driveway of Schultz's home on du Pont's estate in Newtown Square, Pennsylvania. At the time of the shooting, Schultz's wife, Nancy, and du Pont's head of security, Patrick Goodale, were present and witnessed the incident. Goodale was in the passenger seat of du Pont's car when du Pont fired three bullets at Schultz.

After the shooting, du Pont barricaded himself inside his mansion for two days as he negotiated with the police over the phone. The police eventually shut off the power to the house. They were able to capture du Pont when he went outside to fix a heater.

In September 1996, mental health experts ruled du Pont incompetent to stand trial. They testified that he was psychotic and unable to participate in his own defense. He was then committed to a mental hospital for evaluation. During the trial, a psychiatric witness described du Pont as a paranoid schizophrenic. He claimed that du Pont believed there was an international conspiracy to kill him, and he felt threatened by people around him.

Du Pont pleaded not guilty by reason of insanity. However, this defense was rejected by the court. On February 25, 1997, a jury found him guilty of third-degree murder but ruled him mentally ill. Third-degree murder indicates a lack of intent to kill. The judge sentenced du Pont to 13 to 30 years in prison.

After the verdict, Nancy Schultz filed a wrongful death lawsuit against du Pont. The settlement amount was not disclosed, but some reports indicated du Pont would pay at least $35 million.

Du Pont's attorneys filed appeals in the criminal case. In 2000, his case reached the U.S. Supreme Court, which upheld the guilty verdict. He was first eligible for parole on January 29, 2009, but it was denied.

Du Pont died in prison on December 9, 2010, at the age of 72 from chronic obstructive pulmonary disease. He was found unresponsive in his bed and pronounced dead later that morning. Following his will, he was buried in his Foxcatcher wrestling singlet at the Du Pont de Nemours Cemetery in Wilmington, Delaware.