Cholesterol is an important lipid component of cellular membranes. Recent studies have shown that changes in cellular cholesterol level can affect cellular functions. Here, we summarize our recent findings on the impact of cholesterol on the glucose-stimulated Ca2+ signaling in rat pancreatic β cells and the fusion pore kinetics of large dense core granules in rat chromaffin cells. In mouse pancreatic β cells, pharmacological elevation of cellular cholesterol (but not cholesterol extraction) reduced the current density of the delayed rectifier K+ channels, the ATP-dependent K+ channels, and voltage-gated Ca2+ channels. Importantly, cholesterol enrichment impaired glucose-stimulated Ca2+ signaling in mouse pancreatic β cells via a suppression of voltage-gated Ca2+ channels and a decrease in mitochondrial ATP production, which in turn led to a reduction in the glucose-evoked depolarization. In rat chromaffin cells, we found that the persistence of the semi-stable fusion pore was increased by cholesterol enrichment, and acute cholesterol extraction from the cytosolic side of the cell destabilized the semi-stable fusion pore. Overall, our findings highlight the importance of cholesterol in the regulation of cellular signaling and exocytosis.