John Chapman was born on September 26, 1774, in Leominster, Massachusetts, the second child (after his sister Elizabeth) of Nathaniel and Elizabeth Chapman (née Simonds, married February 8, 1770) of Massachusetts. His birthplace has a granite marker, and the street is called Johnny Appleseed Lane.

While Nathaniel was in military Service, his wife died (July 18, 1776) shortly after giving birth to a second son. Chapman ended his military Service and returned home in 1780 to Longmeadow, Massachusetts. In the summer of 1780 he married Lucy Cooley of Longmeadow, Massachusetts, and they had 10 children.

There are stories of Johnny Appleseed practicing his nurseryman craft in the area of Wilkes-Barre, Pennsylvania, and of picking seeds from the pomace at Potomac cider mills in the late 1790s. Another story has Chapman living in Pittsburgh on Grant's Hill in 1794 at the time of the Whiskey Rebellion.

According to some accounts, an 18-year-old John persuaded his 11-year-old brother Nathaniel to go west with him in 1792. The duo apparently lived a nomadic life until their Father brought his large family west in 1805 and met up with them in Ohio. The younger Nathaniel decided to stay and help their Father farm the land.

He cared very deeply about animals, including insects. Henry Howe visited all the counties in Ohio in the early nineteenth century and collected several stories from the 1830s, when Johnny Appleseed was still alive:

The financial panic of 1837 took a toll on his estate. Trees brought only two or three cents each, as opposed to the "fippenny bit" (about six and a quarter cents) that he usually got. Some of his land was sold for taxes following his death, and litigation used up much of the rest.

The Fort Wayne Sentinel printed his obituary on March 22, 1845, saying that he died on March 18:

The Worth family attended First Baptist Church in Fort Wayne, according to records at ACPL, which has one of the nation's top genealogy collections. According to an 1858 interview with Richard Worth Jr., Chapman was buried "respectably" in the Archer cemetery, and Fortriede believes that use of the term "respectably" indicates that Chapman was buried in the hallowed ground of Archer cemetery instead of near the cabin where he died.

John H. Archer, grandson of David Archer, wrote in a letter dated October 4, 1900:

The Johnny Appleseed Commission Council of the City of Fort Wayne reported, "[A]s a part of the celebration of Indiana's 100th birthday in 1916 an iron fence was placed in the Archer graveyard by the Horticulture Society of Indiana setting off the grave of Johnny Appleseed. At that time, there were men living who had attended the funeral of Johnny Appleseed. Direct and accurate evidence was available then. There was little or no reason for them to make a mistake about the location of this grave. They located the grave in the Archer burying ground."

One of the more successful films was Melody Time, the animated 1948 film from Walt Disney Studios featuring Dennis Day. The Legend of Johnny Appleseed, a 19-minute segment, tells the story of an apple farmer who sees others going west, wistfully wishing he was not tied down by his orchard, until an angel appears, singing an apple song, setting Johnny on a mission. When he treats a skunk kindly, all animals everywhere thereafter trust him. The cartoon featured lively tunes, and a childlike simplicity of message. This animated short was included in Disney's American Legends, a compilation of four animated shorts.

From 1962 to 1980, a high school athletic league made up of schools from around the Mansfield, Ohio, area was named the Johnny Appleseed Conference.

In 1966, the U.S. Postal Service issued a 5-cent stamp commemorating Johnny Appleseed.

Fort Wayne, Indiana, is the location of Johnny Appleseed's death. A memorial in Fort Wayne's Swinney Park purports to honor him but not to mark his grave. In Fort Wayne, since 1975, the Johnny Appleseed Festival has been held the third full weekend in September in Johnny Appleseed Park and Archer Park. Musicians, demonstrators, and vendors dress in early 19th century attire and offer food and beverages that would have been available then. In 2008 the Fort Wayne Wizards, a minor league baseball club, changed their name to the Fort Wayne TinCaps. The first season with the new name was in 2009. That same year the Tincaps won their only league championship. The name "Tincaps" is a reference to the tin hat (or pot) Johnny Appleseed is said to have worn. Their team mascot is also named "Johnny."

Steven Fortriede, Director of the Allen County Public Library (ACPL) and author of the 1978 Johnny Appleseed, believes that another gravesite is the correct site, in Johnny Appleseed Park in Fort Wayne,. Johnny Appleseed Park is a Fort Wayne city park that adjoins Archer Park, an Allen County park. Archer Park is the site of John Chapman's grave marker and used to be a part of the Archer family farm.

Shelley Duvall's Tall Tales & Legends featured Johnny Appleseed, as played by Martin Short, in 1986. Also featuring Rob Reiner as Jack Smith and Molly Ringwald as his niece Jenny, the story—while entertaining—takes considerable liberties with the original tall tale.

Johnny Appleseed Elementary School is a public school in Leominster, Massachusetts, his birthplace. Mansfield, Ohio, one of Appleseed's stops in his peregrinations, was home to Johnny Appleseed Middle School until it closed in 1989.

John Clute's science fiction novel Appleseed (2001) centers on a character who may (or may not) be the immortal John Chapman.

In 2003, North Carolina Playwright Keith Smith wrote a one-act musical play titled My Name is Johnny Appleseed, which is presented to school children to show that the true story of John Chapman is just as interesting as the mythical figure, who is shrouded in legend and fable.

Urbana University, in Urbana, Ohio, maintains one of two Johnny Appleseed Museums in the world, which is open to the public. The Johnny Appleseed Educational Center and Museum hosts a number of artifacts, including a tree that is believed to have been planted by Johnny Appleseed. They also provide a number of services for research, including a national registry of Johnny Appleseed's relatives. In 2011 the museum was renovated and updated. The educational center and museum was founded on the belief that those who have the opportunity to study the life of Johnny Appleseed will share his appreciation of education, our country, the environment, peace, moral integrity and leadership.

The village of Lisbon, Ohio, hosts an annual Johnny Appleseed festival September 18–19.

He would tell stories to children and spread The New Church gospel to the adults, receiving a floor to sleep on for the night, and sometimes supper, in return. "We can hear him read now, just as he did that summer day, when we were busy quilting upstairs, and he lay near the door, his voice rising denunciatory and thrillin—strong and loud as the roar of wind and waves, then soft and soothing as the balmy airs that quivered the morning-glory leaves about his gray beard. His was a strange eloquence at times, and he was undoubtedly a man of genius," reported a lady who knew him in his later years. He made several trips back east, both to visit his sister and to replenish his supply of Swedenborgian literature.

In 2016, the history podcast, The Broadsides, did an episode detailing the life and myths surrounding Johnny Appleseed.

Robert A. Heinlein's science fiction novel Farmer in the Sky, which depicts Future colonists on Ganymede and takes up consciously many of the themes of the 19th century American frontier and homesteading, also includes a character who is known as "Johnny Appleseed" and, like the historical one, is involved in planting and spreading apple trees.