Uropathogenic Escherichia coli (UPEC) cause bladder and kidney infections in humans and mice. UPEC initiate many kidney infections by ascending out of infected bladders, but how this occurs is not well understood. To determine if the flagella were responsible for the ascension of UPEC to the kidneys, a fliC mutation in strain NU149 was created. The fliC mutant spread poorly on soft agar plates, and 12h post-inoculation of murine urinary tracts, ascension into the murine kidneys was compromised in this mutant strain compared with wild-type bacteria. Complementation of the mutation restored the ability to spread on soft agar plates and ascend into the murine kidneys. To confirm the fliC mutant results, an anti-flagella monoclonal antibody that has been previously described inhibited the spread of UPEC strain NU149 on soft agar plates. When the anti-flagella antibody was mixed with strain NU149 cells and the antibody-treated bacterial cells were used to infect mice, significantly fewer mice had kidney infections than mice that were injected with strain NU149 cells mixed with normal mouse serum or anti-type 1 pili antibody. These results suggest that E. coli flagella may be of importance in allowing the bacteria to ascend from the bladder and initiate kidney infections in humans, and the use of an antibody against the flagella could prevent the spread of UPEC into the kidneys.