The effects of an acyl-CoA:cholesterol O-acyltransferase (ACAT) inhibitor, N-(3,5-dimethoxy-4-n-octyloxycinnamoyl)-N′-(3,4-dimethylphenyl)piperazine (YIC-C8-434), on cholesterol esterification in the intestine and liver were investigated in vitro and in vivo. YIC-C8-434 inhibited the formation of cholesteryl [³H]oleate from [³H]oleic acid and cholesterol both in human colon adenocarcinoma Caco2 cells and in human hepatoma HepG2 cells with IC₅₀ values of 0.38 and 0.49 μM, respectively. However, it did not influence the incorporation of [³H]oleic acid into triacylglycerols and phospholipids. Oral administration of YIC-C8-434 at a dose of 8.3 mg/kg/d inhibited [¹⁴C]cholesterol absorption by 17% (p<0.01) in rats. YIC-C8-434 also significantly reduced the secretion of very low-density lipoprotein (VLDL) cholesterol from the liver into the plasma at an oral dose of 100 mg/kg/d after an intravenous injection of Triton WR-1339. These results suggest that oral administration of YIC-C8-434 reduces intestinal cholesterol absorption and hepatic VLDL cholesterol secretion by direct inhibition of ACAT in the intestinal epithelium and hepatocytes, respectively. However, the inhibitory action of YIC-C8-434 on cholesterol absorption rather than hepatic cholesterol secretion may play a more important role in its hypocholesterolemic activity, because the effective dose for the former was 12-fold lower than that for the latter.