Abstract Acyl-coenzyme A:cholesterol O-acyltransferase-1 (ACAT-1) plays an essential role in macrophage foam cell formation and progression of atherosclerosis. We developed a potent and selective ACAT-1 inhibitor, K-604, and tested its effects in apoE-knockout mice. Administration of K-604 to 8-week-old apoE-knockout mice for 12 weeks at a dose of 60 mg/kg/day significantly reduced macrophage-positive area and increased collagen-positive area in atherosclerotic plaques in the aorta without affecting plasma cholesterol levels or lesion areas, indicating direct plaque-modulating effects of K-604 on vascular walls independent of plasma cholesterol levels. Pactimibe, a nonselective inhibitor of ACAT-1 and ACAT-2, reduced plasma cholesterol levels but did not affect macrophage- or collagen-positive areas. The size of macrophages and cholesteryl ester contents in the aorta were reduced by K-604. Exposure of cultured human aortic smooth muscle cells to K-604 resulted in increased procollagen type 1 contents in the culture supernatant and increased procollagen type 1 mRNA levels. Procollagen production was unaffected by pactimibe even at a concentration that inhibited cholesterol esterification to the basal level. Thus, the plaque-modulating effects of K-604 can be explained by stimulation of procollagen production independent of ACAT inhibition in addition to potent inhibition of macrophage ACAT-1.