Recent studies have demonstrated that cholesterol elevation in pancreatic islets is associated with a reduction in glucose-stimulated insulin secretion, but the underlying cellular mechanisms remain elusive. Here, we show that cholesterol enrichment dramatically reduced the proportion of mouse β-cells that exhibited a Ca(2+) signal when stimulated by high glucose. When cholesterol-enriched β-cells were challenged with tolbutamide, there was a decrease in the amplitude of the Ca(2+) signal, and it was associated with a reduction in the cell current density of voltage-gated Ca(2+) channels (VGCC). Although the cell current densities of the ATP-dependent K(+) channels and the delayed rectifier K(+) channels were also reduced in the cholesterol-enriched β-cells, glucose evoked only a small depolarization in these cells. In cholesterol-enriched cells, the glucose-mediated increase in cellular ATP content was dramatically reduced, and this was related to a decrease in glucose uptake via glucose transporter 2 and an impairment of mitochondrial metabolism. Thus, cholesterol enrichment impaired glucose-stimulated Ca(2+) signaling in β-cells via two mechanisms: a decrease in the current density of VGCC and a reduction in glucose-stimulated mitochondrial ATP production, which in turn led to a smaller glucose-evoked depolarization. The decrease in VGCC-mediated extracellular Ca(2+) influx in cholesterol-enriched β-cells was associated with a reduction in the amount of exocytosis. Our findings suggest that defect in glucose-stimulated Ca(2+) signaling is an important mechanism underlying the impairment of glucose-stimulated insulin secretion in islets with elevated cholesterol level.