Alteration of pancreatic -cell survival and Preproinsulin gene expression by prolonged hyperglycemia may result from increased c-MYC expression. However, it is unclear whether c-MYC effects on -cell function are compatible with its proposed role in glucotoxicity. We therefore tested the effects of short-term c-MYC activation on key -cell stimulus-secretion coupling events in islets isolated from mice expressing a tamoxifen-switchable form of c-MYC in -cells (MycER) and their wild-type littermates. Tamoxifen treatment of wild-type islets did not affect their cell survival, Preproinsulin gene expression and glucose stimulus-secretion coupling. In contrast, tamoxifen-mediated c-MYC activation for 2-3 days triggered cell apoptosis and decreased Preproinsulin gene expression in MycER islets. These effects were accompanied by mitochondrial membrane hyperpolarization at all glucose concentrations, a higher resting intracellular calcium concentration ([Ca²⁺]_i), and lower glucose-induced [Ca²⁺]_i rise and islet insulin content leading to a strong reduction of glucose-induced insulin secretion. Compared with these effects, 1 week culture in 30 mmol.l^-1 glucose increased the islet sensitivity to glucose stimulation without reducing the maximal glucose effectiveness nor the insulin content. In contrast, overnight exposure to a low H₂O₂ concentration increased the islet resting [Ca²⁺]_i and reduced the amplitude of the maximal glucose response as in tamoxifen-treated MycER islets. In conclusion, c-MYC activation rapidly stimulates apoptosis, reduces Preproinsulin gene expression and insulin content, and triggers functional alterations of -cells that are better mimicked by overnight exposure to a low H₂O₂ concentration than by prolonged culture in high glucose.