Diabetes mellitus is a systemic disease associated with a deficiency of insulin production or action. Diabetic patients have an increased susceptibility to infection with the urinary tract being the most common site. Recent studies suggest that Ribonuclease 7 (RNase 7) is a potent antimicrobial peptide that plays an important role in protecting the urinary tract from bacterial insult. Because the impact of diabetes on RNase 7 expression and function are unknown, we investigated the effects of insulin on RNase 7 using human urine specimens. The urinary RNase 7 concentrations were measured in healthy control patients and insulin-deficient type 1 diabetics before and after starting insulin therapy. Compared with controls, diabetic patients had suppressed urinary RNase 7 concentrations, which increased with insulin. Using primary human urothelial cells, the mechanisms by which insulin stimulates RNase 7 synthesis were next explored. Insulin induced RNase 7 production via the phosphatidylinositide 3-kinase signaling pathway (PI3K/AKT) to shield urothelial cells from uropathogenic E. coli. In contrast, uropathogenic E. coli suppressed PI3K/AKT activity and RNase 7 production. Thus, insulin and PI3K/AKT signaling are essential for RNase 7 expression and increased infection risks in diabetic patients may be secondary to suppressed RNase 7 production. Our data may provide unique insight into novel urinary tract infection therapeutic strategies in at-risk populations.