The pancreatic β cell can respond in the long term to hyperglycemia both with an increased capacity for insulin production and, in susceptible individuals, with apoptosis. When glucose-induced apoptosis offsets the increasing β cell capacity, type 2 diabetes results. Here, we tested the idea that the pathway of glucose metabolism that leads to the modification of intracellular proteins with the O-linked monosaccharide N-acetylglucosamine (O-Glc-NAc) is involved in the glucose-induced apoptosis. This idea is based on two recent observations. First, the β cell expresses much more O-GlcNAc transferase than any other known cell, and second, that the β cell-specific toxin, streptozotocin (STZ), itself a GlcNAc analog, specifically blocks the enzyme that cleaves O-GlcNAc from intracellular proteins. As a consequence, we now show that hyperglycemia leads to the rapid and reversible accumulation of O-GlcNAc specifically in β cells in vivo. Animals pretreated with STZ also accumulate O-GlcNAc in their β cells when hyperglycemic, but this change is sustained upon re-establishment of euglycemia. In concert with the idea that STZ toxicity results from the sustained accumulation of O-GlcNAc after a hyperglycemic episode, we established a low-dose STZ protocol in which the β cells' toxicity of STZ was manifest only after glucose or glucosamine administration. Transgenic mice with impaired β cell glucosamine synthesis treated with this protocol are resistant to the diabetogenic effect of STZ plus glucose yet succumb to STZ plus glucosamine. This study provides a causal link between apoptosis in β cells and glucose metabolism through glucosamine to O-GlcNAc, implicating this pathway of glucose metabolism with β cell glucose toxicity.