Objective. An increased glomerular filtration rate (GFR) has been postulated as a potential mechanism involved in the progression of diabetic nephropathy. Studies suggest that C-peptide exerts a reno-protective effect on diabetes. The peptide decreases hyperfiltration in patients with Type 1 diabetes, as well as in diabetic animal models. In this study, we investigated whether C-peptide causes a change in arteriolar diameter. Research Design and Methods. C57-Bl mice were made diabetic by means of a single i.v. injection of alloxan two weeks prior to the experiment. Age-matched normoglycemic mice served as controls. Afferent arterioles, intact with the glomeruli, were dissected and microperfused. The effect of luminal application of C-peptide, as compared to scrambled C-peptide or vehicle, was investigated. The effect of the Rho-kinase inhibitor Y-27632 was also investigated. Results. C-peptide constricted afferent arterioles in diabetic mice by -27 % when compared to the control value. Normoglycemic arterioles administered C-peptide, displayed a delayed and minute response (-4 %). Scrambled C-peptide or vehicle administration, whether administered to hyperglycemic or normoglycemic mice, did not induce any effect. Addition of Y-27632 abolished the effect of C-peptide. Conclusion. C-peptide induces constriction of afferent arterioles in diabetic mice. This can reduce enhanced GFR, and may be one of the mechanisms in the reno-protective action of C-peptide in diabetes.