Abstract: Objective To investigate the effects of nicotinamide on blood glucose control in gestational diabetes mellitus (GDM) rats by detecting the mitochondrial superoxide level and anti-oxidative enzymes. Methods Diabetes Mellitus (GDM) rats were administered nicotinamide at daily doses of 50, 100 or 200 mg/kg body weight by oral gavage from gestation days 6 through 20. Blood glucose level was measured on gestation day 21. Brain tissue of the fetus rat and skeletal muscle of GDM rats in each group were collected. The expression and activity of anti-oxidative enzymes, including SOD1, SOD2 and Catalase were measured by RT-PCR and Western blot. Results Nicotinamide significantly lowered the blood glucose in GDM rats and decreased mitochondrial superoxide level in the fetus neurons. Superoxide dismutase 2 (SOD2) was induced by nicotinamide in GDM rats (P<0.05). Nicotinamide also promoted SIRT3 activity and deacetylated SOD2 in GDM rats (P<0.05), which enhanced SOD2 activity and altered mitochondrial ROS. Conclusion Nicotinamide promoted SIRT3 activity to deacetylate SOD2, which activated SOD2 and affected the mitochondrial ROS in GDM rats. Therefore, nicotinamide could potentially treat GDM patients in clinical application.