Although TLR5 regulates the innate immune response to bacterial flagellin, it is unclear whether its function is essential during in vivo murine infections. To examine this question, we challenged Tlr5(-/-) mice transurethrally with Escherichia coli. At 2 days postinfection, wild-type mice exhibited increased inflammation of the bladder in comparison to Tlr5(-/-) mice. By day 5 postinfection, Tlr5(-/-) mice had significantly more bacteria in the bladders and kidneys in comparison to wild-type mice and showed increased inflammation in both organs. In addition, flagellin induced high levels of cytokine and chemokine expression in the bladder that was dependent on TLR5. Together, these data represent the first evidence that TLR5 regulates the innate immune response in the urinary tract and is essential for an effective murine in vivo immune response to an extracellular pathogen.