We studied the role of cholesterol in regulated protein secretion in neuroendocrine cells by manipulating the cholesterol content of AtT‐20 cells. Depletion of cellular cholesterol levels caused a reversible block of immature secretory granule biogenesis at the level of the trans‐Golgi‐network, whereas increased cholesterol levels promoted immature secretory granule formation. Cholesterol depletion also blocked the formation of constitutive secretory vesicles, but did not inhibit the transport between the endoplasmic reticulum and the Golgi complex. Our results indicate that the assembly of cholesterol‐based lipid microdomains is required for the biogenesis of both regulated and constitutive secretory vesicles from the trans‐Golgi‐network in neuroendocrine cells.