In a post on Facebook, authorities said the vehicle was stolen on Wednesday afternoon from the Strawberry Festival Grounds in Plant City, about 60 miles from Orlando.
Authorities have not said how many doses of the vaccine were inside the car.
The vaccine was intended for distribution at the Plant City vaccination center on the festival grounds, CNN affiliate WKMG reported. The stolen vehicle is described as a 2018 gray Hyundai Accent with the Florida tag NPJJ58.
Police released surveillance video of a man they would like to question in connection with the theft.
Authorities are asking that anyone with information about the theft contact the Plant City Police Department.