Streptozotocin injection in animals destroys pancreatic β cells, leading to insulinopenic diabetes. Here, we evaluated the toxic effect of streptozotocin (STZ) in GLUT2^−/− mice reexpressing either GLUT1 or GLUT2 in their β cells under the rat insulin promoter (RIPG1 × G2^−/− and RIPG2 × G2^−/− mice, respectively). We demonstrated that injection of STZ into RIPG2 × G2^−/− mice induced hyperglycemia (>20 mM) and an ∼80% reduction in pancreatic insulin content. In vitro, the viability of RIPG2 × G2^−/− islets was also strongly reduced. In contrast, STZ did not induce hyperglycemia in RIPG1 × G2^−/− mice and did not reduce pancreatic insulin content. The viability of in vitro cultured RIPG1 × G2^−/− islets was also unaffected by STZ. As islets from each type of transgenic mice were functionally indistinguishable, these data strongly support the notion that STZ toxicity toward β cells depends on the expression of GLUT2.