When Europeans made first contact, the Potano lived in the area, but by 1685, northern tribes supplied with arms and often joined by English settlers from the Province of Carolina, were raiding Potano territory, burning villages, killing some Potano and carrying others away to be sold as slaves in Charles Towne. These raids continued into the 18th century and eventually led to the extinction of the tribe.

Gainesville was established in 1854 and named after Edmund P. Gaines, a career United States Army officer who served for nearly fifty years, and attained the rank of major general. One of the Army's senior commanders during its formative years, he was a veteran of the War of 1812, Seminole Wars, Black Hawk War, and Mexican-American War.

Gainesville has been recognized by the National Arbor Day Foundation every year since 1982 as a "Tree City, USA" and was recognized in 2020 as a "tree city of the world". Its tree canopy is both dense and species rich, including broadleaf evergreens, conifers, and deciduous species. And a 2016 ecological assessment determined that Gainesville's urban tree canopy covers 47 percent of its total land area.

The University of the State of Florida was moved from Lake City to Gainesville in 1906 and its name was simplified to University of Florida in 1909.

Albert Einstein Gator and Alberta Gator are the official mascots of the University of Florida. The alligator was chosen as the school mascot in 1911, after a local vendor ordered and sold school pennants imprinted with an alligator since the animal is common in the Gainesville area and throughout the state. The mascot was a popular choice, and the university's sports teams quickly adopted the Gator nickname.

The sports drink Gatorade was invented in Gainesville in the 1960s to help refresh the University of Florida football team. UF still receives a share of the profits from the beverage, but Gatorade's headquarters are now in Chicago.

The otherwise nondescript poured concrete wall is notable for being almost entirely covered by graffiti. It has served as a sort of ever-changing bulletin board for the Gainesville community since the 1980s. Items painted on the wall have included marriage proposals, birthday wishes, graduation announcements, celebration of athletic victories, activist and public awareness statements, tributes, promotions, and memorials. After decades of use, it is estimated that paint on the center of the wall is about 250 layers thick.

In April 2003, Gainesville became known as the "Healthiest Community in America" when it won the only "Gold Well City" award given by the Wellness Councils of America (WELCOA). As of July 2011, Gainesville remained the only city in the country to win the award.

On September 21, 2006, Tom Petty and the Heartbreakers received the keys to the city of Gainesville, Florida, where the band was formed in 1976, and where most of the bandmembers grew up. When questioned about the key he received from Gainesville's mayor, Petty quipped, "It's a lot nicer than the one we got in Chicago."

Although built to resemble a strand of DNA, the structure is not a true double helix, but instead a pair of connected sine waves 180 degrees out of phase. At night, the bridge is lit by an array of LED lights.