CD38 is a multifunctional enzyme capable of generating metabolites that release Ca2+ from intracellular stores, including nicotinic acid adenine dinucleotide phosphate (NAADP). A number of studies have led to the controversial proposal that CD38 mediates an alternate pathway for glucose-stimulated insulin release and contributes to the pathogenesis of diabetes. It has recently been shown that NAADP mediates Ca2+ mobilization by insulin in human pancreatic β-cells. In the present study, we report altered Ca2+ homeostasis and reduced responsiveness to insulin, but not glucose, in Cd38−/− β-cells. In keeping with the antiapoptotic role of insulin signaling, Cd38−/− islets were significantly more susceptible to apoptosis compared with islets isolated from littermate controls. This finding correlated with disrupted islet architecture and reduced β-cell mass in Cd38−/− mice, both in the context of a normal lab diet and a high-fat diet. Nevertheless, we did not find robust differences in glucose homeostasis in vivo or glucose signaling in vitro in Cd38−/− mice on the C57BL/6 genetic background, in contrast to previous studies by others of Cd38 knockout mice on the ICR background. Thus, our results suggest that CD38 plays a role in novel antiapoptotic signaling pathways but does not directly control glucose signaling in pancreatic β-cells.