Protein expression profiles in rat bladder smooth muscle were compared between animal models of STZ-induced diabetes mellitus (STZ-DM) and age matched controls (AMC) at one week and two months after induction of hyperglycemia with STZ treatment. At each time point, protein samples from four STZ-DM and four AMC rat bladder tissues were prepared independently and analyzed together across multiple DIGE gels using a pooled internal standard sample to quantify expression changes with statistical confidence. A total of 100 spots were determined to be significantly changing among the four experimental groups. A subsequent mass spectrometry analysis of the 100 spots identified a total of 56 unique proteins. Of the proteins identified by 2D-DIGE/MS, 10 exhibited significant changes one week after STZ-induced hyperglycemia while the rest showed differential expression after two months. A network analysis of these proteins using MetacoreTM suggested induction of transcriptional factors that are too low to be detected by 2D-DIGE and identified an enriched cluster of down regulated proteins that are involved in cell adhesion, cell shape control and motility; including vinculin, intermediate filaments, Ppp2r1a, and extra cellular matrix (ECM) proteins. The proteins that are up-regulated include proteins involved in muscle contraction (eg., Mrlcb, and Ly-GDI), in glycolysis (eg., -enolase, and Taldo1), in mRNA processing (eg., hnRNP A2/B1), in inflammatory response (eg., S-100A9, Anexin1, and ApoA-I), and in chromosome segregation and migration (eg., Tuba1, and Vil2). Our results suggest that the development of diabetes related complications in this model involves the down regulation of structural and ECM proteins in smooth muscle that are essential for the normal muscle contraction and relaxation but also induces proteins that are associated with cell proliferation and inflammation that may account for some of the functional deficits known to occur in diabetic complications of bladder.