The major thoroughfare Interstate 75 passes a few miles to the south of downtown Gainesville, running from Miami at one end to the Canadian border at Sault Sainte Marie (north of Detroit) and goes through Cincinnati, Lexington (Kentucky), Atlanta and Tampa along the way. There are also several state highways (20, 24, 25, 26) and a few federal ones (441, 301). The city is laid out in more or less a grid fashion and sensibly named, with all named avenues, places, roads and lanes running east-west and all other street suffixes denoting north-south. Driving, therefore, is a good way to get about the city. There are several car rental locations (Enterprise, Avis, Budget, and others) within Gainesville if you do not get a car at the airport or come with your own.

The city also has a bus-based Regional Transit System (RTS) that runs throughout the city as well as throughout the University of Florida. Buses for each line run anywhere from once every 15 minutes to once every hour. Routes are wheelchair accessible and service all parts of the urban center; most buses also come with bike racks at the front available for use, free of extra charge. Fare is $1 for adults and $.50 for seniors or students.