During the 1920s and 1930s, the couple acquired more land and developed Liseter Hall Farm for Thoroughbred breeding, showing, and racing. His mother retained Liseter Hall Farm after the couple divorced in 1941. She added a dairy herd of Guernseys and bred Welsh ponies at the farm. John was 2 when his parents divorced. He had two older sisters, Jean and Evelyn, an older brother Henry E. I. du Pont, and a younger half brother, william du Pont III, born of their father's second marriage.

John du Pont was born on November 22, 1938, in Philadelphia, Pennsylvania, the youngest of four children of william du Pont, Jr. and Jean Liseter Austin (1897–1988). He grew up at Liseter Hall, a mansion built in 1922 in Newtown Square, Pennsylvania, by his maternal grandfather on more than 200 acres of land given to his parents at their wedding by his maternal grandfather. Both his parents' families had emigrated from Europe to the United States at the beginning of the 19th century and became highly successful.

Du Pont founded the Delaware Museum of Natural History in 1957, which opened to the public in 1972 on a site near Winterthur donated by his relative Henry Francis du Pont. John du Pont served on the board for many years. He also helped fund a new basketball arena at Villanova University, which opened in 1986. Originally it was called the John Eleuthère du Pont Pavilion, but after his conviction, his name was removed from the facility. Today it is called The Pavilion.

In 1972, du Pont founded and directed the Delaware Museum of Natural History and contributed to Villanova University and other institutions.

Du Pont's friends said the shooting was uncharacteristic. Joy Hansen Leutner, a Triathlete from Hermosa Beach, California, lived for two years on the estate. Leutner said du Pont helped her through a stressful period in the mid 1980s. She later said, "With my family and friends, John gave me a new lease on life. He gave more than money; he gave himself emotionally." She expressed incredulity about the killing. She is quoted as saying: "There's no way John in his right mind would have killed Dave." Newtown Township supervisor John S. Custer Jr. said, "At the time of the murder, John didn't know what he was doing." Charles King, Sr., a du Pont stable hand and manager for 30 years, claimed he knew du Pont well throughout his life. King's son, Charles "Chuckie" King Jr., said he considered du Pont his friend during his childhood.

At the age of 45, on September 3, 1983, du Pont married 29-year-old Gale Wenk, an occupational therapist. They met after he injured his hand in an auto accident. They lived together for less than six months. Du Pont filed for divorce when they had been married for ten months. Wenk sued du Pont for $5 million, claiming he had pointed a gun at her and tried to push her into a fireplace. The divorce became final in 1987. Du Pont's will excluded her from inheriting any of his estate. In 1987, it was estimated that John du Pont was worth $200 million.

Du Pont had been worth an estimated US $200 million in 1986, about $450 million in current dollars. His will bequeathed 80 percent of his estate to Bulgarian Wrestler Valentin Yordanov, an Olympic champion who had trained at Foxcatcher, and Yordanov's relatives. In June 2011, du Pont's niece Beverly A. du Pont Gauggel and nephew william H. du Pont filed a petition to challenge the will in Media, Pennsylvania, asserting that du Pont was not "of sound mind" when he made his will. The petition claims that during that period, John du Pont asserted alternately that he was Jesus Christ, the Dalai Lama, and a Russian tsar.

After his mother's death in 1988, du Pont assumed stewardship of Liseter Farm and renamed it "Foxcatcher Farm" after his father's famed Thoroughbred racing stable. John never lived in the manor house; he occupied a smaller house on the estate. Days after his mother's death, he moved into the main house. He maintained much of her work, but added a wrestling facility and supporting buildings for that interest.

In the 1990s, friends and acquaintances were concerned about his erratic and paranoid behavior, but his wealth shielded him.

Du Pont became a sponsor in wrestling, swimming, track, and the modern pentathlon. He was also involved in promoting a subset of the modern pentathlon (run, swim, shoot) as a separate event. He took up athletics and became a competitive Wrestler in his 50s. His only prior wrestling experience was as a freshman in high school. He began competing again at the age of 55 in the 1992 Veteran's World Championships in Cali, Colombia; following that in 1993 in Toronto, Ontario; in 1994 in Rome, Italy; and in 1995 in Sofia, Bulgaria.

After his arrest, du Pont sold off the dairy herd, nearly 70 Guernseys, in the fall of 1996. He ordered all the buildings at Foxcatcher Farm to be painted a matte black. The Delaware Museum of Natural History, which du Pont formerly headed and which held the dairy farm in trust, sold that portion in January 1998 after his conviction and sentencing to prison. A 123-acre segment is now occupied by the campus of The Episcopal Academy, a private independent K–12 school founded in 1785, which moved there in 2008 from split campuses located in the nearby Philadelphia Main Line communities of Merion and Devon. The 90-year-old du Pont mansion, Liseter Hall, in which du Pont was raised and had lived for 57 years, was demolished by Glen Miller Demolition in January 2013. The mansion stood on a 400-acre portion of the property that is now being developed by Toll Brothers into a "master planned community of 449 luxury homes" called "Liseter Estate." Most of the outbuildings were torn down, though an existing 7,000 square-foot historical barn will be used as a clubhouse in the new development.

Du Pont pleaded "not guilty by reason of insanity." The insanity defense was thrown out by the court, and on February 25, 1997, a jury found him guilty of third-degree murder but mentally ill. In Pennsylvania, third-degree murder is a lesser charge than first-degree (intentional) or second-degree (during the perpetration of a felony), and indicates a lack of intent to kill. In Pennsylvania Criminal code, "insanity" applies to someone whose "disease or defect" leaves him unable either to understand that his conduct is wrong or to conform it to the law.

Du Pont's attorneys filed appeals. In 2000, his case reached the U.S. Supreme Court, which upheld the verdict. Du Pont was first eligible for parole on January 29, 2009; it was denied. Du Pont's maximum sentence would have ended on January 29, 2026, when he would have been 87. In 2010, the 3rd Circuit U.S. appeals court in Philadelphia rejected all but one issue raised on appeal (involving his use of a Bulgarian prescription drug, scopolamine, before he fatally shot Schultz in 1996), and requested written briefs.

Du Pont died at the age of 72 on December 9, 2010, from chronic obstructive pulmonary disease (COPD). A spokesperson for the Pennsylvania Department of Corrections said du Pont was found unresponsive in his bed at the State Correctional Institution – Laurel Highlands. He was pronounced dead at 6:55 a.m. at Somerset Community Hospital. He was buried in his red Foxcatcher wrestling singlet, in accordance with his will, at the Du Pont de Nemours Cemetery in Wilmington, Delaware.

That petition was dismissed, and while appealed, the Superior Court of Pennsylvania has upheld a Delaware County Orphans Court order dismissing a challenge on the will on November 19, 2012. Former Delaware County Court of Common Pleas President Judge Joseph Cronin dismissed the challenge for lack of standing, finding that because the niece and nephew were not named in two successive wills going back to 2006, they would not be harmed if the September 2010 will were deemed valid. A three-judge panel of the Superior Court affirmed that ruling on November 19, 2012.

The jury verdict of "guilty but mentally ill" meant the sentencing would be referred to the judge, Patricia Jenkins. She could have sentenced du Pont to 5 to 40 years. Du Pont was sentenced to 13 to 30 years' incarceration and was housed at the State Correctional Institution – Mercer, a minimum-security institution in the Pennsylvania prison system. Du Pont was initially confined to Cresson Correctional Institute. Following the guilty verdict, Nancy Schultz, Dave's widow, filed a wrongful death lawsuit against du Pont. The amount of the settlement was not disclosed. The Philadelphia Inquirer, citing anonymous sources, reported du Pont was to pay Schultz at least $35 million.

Du Pont was also a philatelist. Bidding anonymously in a 1980 auction, he paid $935,000 for one of the rarest stamps in the world, the British Guiana 1856 1c black on magenta. After his death, this stamp was sold at auction for $9.5 million (inclusive of buyer's premium) at Sotheby's June 17, 2014. For the fourth time, the stamp broke the record for a single stamp's sale. The unique stamp was part of the estate of du Pont. According to du Pont's will – unsuccessfully challenged by several parties – 80 percent of the sale proceeds go to the family of Bulgarian Wrestler Valentin Jordanov Dimitrov and 20 percent goes to the Eurasian Pacific Wildlife Foundation, based in Paoli, Pennsylvania, a group du Pont founded to support Pacific wildlife. In 1986, competing as "John Foxbridge", he won the Grand Prix d'Honneur in the FIP Championship Class at the STOCKHOLMIA 86 international stamp exhibition for his display of "British North America". While du Pont continued to buy stamps while in prison, he was not allowed to bring them there.

During an October 2015 podcast, Mark Schultz revealed that when John was about 30 years old, a horse he was riding threw him onto a fence, injuring his testicles. They became infected and were removed, resulting in androgynous characteristics for the remainder of his life.