Recent evidence indicates that antimicrobial peptides (AMPs) serve key roles in defending the urinary tract against invading uropathogens. To date, the individual contribution of AMPs to urinary tract host defense is not well defined. In this study, we identified Regenerating islet-derived 3 gamma (RegIIIγ) as the most transcriptionally up-regulated AMP in murine bladder transcriptomes following uropathogenic Escherichia coli (UPEC) infection. We confirmed induction of RegIIIγ mRNA during cystitis and pyelonephritis by quantitative RT-PCR. Immunoblotting demonstrates increased bladder and urinary RegIIIγ protein levels following UPEC infection. Immunostaining localizes RegIIIγ protein to urothelial cells of infected bladders and kidneys. Human patients with UTI have increased urine concentrations of the orthologous Hepatocarcinoma-Intestine-Pancreas / Pancreatitis Associated Protein (HIP/PAP) compared to healthy controls. Recombinant RegIIIγ protein does not demonstrate bactericidal activity toward UPEC in vitro, but does kill Staphylococcus saprophyticus in a dose-dependent manner. Kidney and bladder tissue from RegIIIγ knockout mice and wild-type mice contain comparable bacterial burden following UPEC and Gram-positive UTI. Our results demonstrate that RegIIIγ and HIP/PAP expression is induced during human and murine UTI. However, their specific function in the urinary tract remains uncertain.