Background: Both C-peptide and nicotinamide are known to reduce blood glucose in type 1 diabetes. In the present study, the effects of C-peptide alone or in combination with nicotinamide on glucose and insulin levels in streptozotocin-nicotinamide-induced type 2 diabetic mice. Methods: The study used 70 adult male NMARI mice, weighing 25–35 g, divided into seven groups: control; type 1 diabetic; type 2 diabetic; type 2 diabetic + C-peptide; type 2 diabetic + nicotinamide; type 2 diabetic + nicotinamide and C-peptide; type 2 diabetic + glyburide. Type 2 diabetes was induced with ip injection of streptozotocin–nicotinamide. Twenty eight days after the onset of diabetes, treatment with C-peptide, nicotinamide, nicotinamide + C-peptide, or glyburide were initiated. Glucose and insulin levels were evaluated. One-way ANOVA and Least Significant Difference (LSD) tests were used to test for significance. Results: Blood glucose significantly increased (P < 0.001) in all diabetic mice compared with control mice. Insulin resistance and blood glucose levels were significantly reduced (P < 0.05) in C-peptide and nicotinamide + C-peptide mice compared with type 2 diabetic mice. Conclusions: The present study supports the anti-diabetic effects of C-peptide, nicotinamide + C-peptide, and suggests that one of the anti-diabetic mechanisms of these compounds is mediated through the reduction of insulin resistance.