Cholesterol is reportedly abundant in the endocrine secretory granule (SG) membrane. In this study, we examined the involvement of cholesterol biosynthesis intermediates and inhibitors in insulin secretion and SG formation mechanisms. There are two routes for the supply of cholesterol to the cells: one via de novo biosynthesis and the other via low-density lipoprotein receptor-mediated endocytosis. We found that insulin secretion and content are diminished by β-hydroxy-β-methylglutaryl-coenzyme A inhibitor lovastatin but not by lipoprotein depletion from the culture medium in MIN6 β-cells. Cholesterol biosynthesis intermediates mevalonate, squalene, and geranylgeranyl pyrophosphate enhanced glucose-stimulated insulin secretion, and the former two increased insulin content. The glucose-stimulated insulin secretion-enhancing effect of geranylgeranyl pyrophosphate was also confirmed in perifusion with rat islets. Morphologically, mevalonate and squalene increased the population of SGs without affecting their size. In contrast, lovastatin increased the SG size with reduction of insulin-accumulating dense cores, leading to a decrease in insulin content. Furthermore, insulin was secreted in a constitutive manner, indicating disruption of regulated insulin secretion. Because secretogranin III, a cholesterol-binding SG-residential granin-family protein, coincides with SG localization based on the cholesterol composition, secretogranin III may be associated with insulin-accumulating mechanisms. Although the SG membrane exhibits a high cholesterol composition, we could not find detergent-resistant membrane regions using a lipid raft-residential protein flotillin and a fluorescent cholesterol-Si-pyrene probe as markers on a sucrose-density gradient fractionation. We suggest that the high cholesterol composition of SG membrane with 40-50 mol% is crucial for insulin secretion and SG formation functions.