Cholesterol is crucial for the survival of cells, and cholesterol and its metabolites are involved in the pathogenesis of atherosclerosis, cancer, neurodegenerative diseases etc. Acyl-CoA:cholesterol acyltransferase (ACAT) catalyzes the esterification of cholesterol in cells, and plays an important role in the intracellular cholesterol homeostasis. ACAT is the therapeutic target of many diseases. This article will review the role of ACAT in cholesterol metabolism and associated diseases.