Cholesterol is essential for neuronal activity and function. Cholesterol depletion in the plasma membrane impairs synaptic transmission. However, the molecular mechanisms by which cholesterol deficiency leads to defects in vesicle fusion remain poorly understood. Here we show that cholesterol is required for Ca2+-dependent fusion using the in-vitro reconstitution of vesicle fusion, atomic force microscopy (AFM), and amperometry to monitor exocytosis in chromaffin cells. Purified native vesicles were crucial for the complete reconstitution of physiological Ca2+-dependent fusion, whereas vesicle-mimicking liposomes failed to reproduce the cholesterol effect. Intriguingly, cholesterol had no effect on membrane insertion of synaptotagmin-1, a Ca2+ sensor for ultrafast fusion. Cholesterol stabilizes local membrane bending induced by synaptotagmin-1 insertion, thereby lowering the energy barrier for Ca2+-dependent fusion to occur. Our data provide evidence that cholesterol depletion abolishes Ca2+-dependent vesicle fusion by disrupting synaptotagmin-1-induced membrane bending, and suggests that cholesterol is a master regulator for Ca2+-dependent fusion.