AbstractAcyl‐coenzyme A:cholesterol acyltransferase (ACAT) 1 and 2 are both involved in intracellular cholesterol esterification. The expression of acyl‐coenzyme A:cholesterol acyltransferase 1; (ACAT1) is ubiquitous, whereas, enterocytes and hepatocytes exclusively express acyl‐coenzyme A:cholesterol acyltransferase 2 (ACAT2). Studies in mice lacking ACAT1 showed extensive deposition of unesterified cholesterol in skin and brain, and larger atherosclerotic lesions compared to controls. ACAT2 deficiency in mice protected against atherosclerosis, diet‐induced hypercholesterolemia and cholesterol gallstone disease, and reduced hepatic lipid accumulation and decreased serum cholesterol levels. Two unspecific ACAT inhibitors have been tested in humans. In the face of the results in ACAT1 deficient mice, strongly arguing against an ACAT1 inhibition, it was not surprising that none of these inhibitors showed beneficial effects on the predefined primary endpoints of the studies. However, and still to be proven, ACAT2 inhibition might be a therapeutic option for the treatment and prevention of cardiovascular disease. In this review, we aim to outline some important aspects on the role of ACAT in cholesterol esterification and to reappraise ACAT2 as therapeutic target.