The remarkable resistance of the urinary tract to infection has been attributed to its physical properties and the innate immune responses triggered by pattern recognition receptors such as TLRs. We report a distinct TLR4-mediated mechanism in bladder epithelial cells (BECs) that abrogates bacterial invasion, a necessary step for successful infection. Compared to controls, uropathogenic type 1 fimbriated Escherichia coli and Klebsiella pneumoniae invaded BECs of TLR4 mutant mice in 10-fold or greater numbers. TLR4-mediated suppression of bacterial invasion was linked to increased intracellular cAMP levels, which negatively impacted Rac-1-mediated mobilization of the cytoskeleton, a critical step in bacterial invasion. Artificially increasing intracellular cAMP levels in BECs of TLR4 mutant mice restored resistance to type 1 fimbriated bacterial invasion. These findings reveal that in addition to their well known function in cytokine signaling, TLRs mediate proximal signaling events to actively inhibit bacterial invasion in the bladder.