In diabetic patients, glucolipotoxicity induces multiple defects in β-cells. Furthermore, increasing evidence also confirms the direct interaction between the adipocytes and β-cells. The beneficial efficacy of vitamin C (Vc) on β-cells is rarely investigated. In this study, INS-1 832/13 β-cells were cultured with high levels of glucose and free fatty acid (FFA) or cocultured with 3T3-L1 adipocytes in the presence or absence of Vc. Vc decreased glucolipotoxicity-induced cell mass loss by reducing apoptosis and reactive oxidative species (ROS). After treatment with elevated glucose and FFA, β-cell secretion dysfunction was evidenced and was partially improved by 1, 10 and 50 μg/mL Vc treatment. In the coculture system, impaired secretion function was also moderately normalized upon addition of 10 and 50 μg/mL Vc to the coculture medium (p<0.05). Vc at 50 μg/mL significantly (p<0.05) inhibited the fatty acid release from adipocytes to the coculture medium. Meanwhile, the elevated ROS of cocultured β-cells was decreased in the presence of Vc (1 to 50 μg/mL). In both induction methods, intracellular TGs in both β-cells and adipocytes were decreased by Vc treatment; however, Vc did not affect the intracellular insulin level. Moreover, IL-6 and adiponectin levels in the coculture medium remained under the levels of the control group. The positive effects of Vc might be due to the antioxidant capacity and TG inhibitory effect of Vc.