Acyl-CoA: cholesterol acyltransferase (ACAT) is a key enzyme for cholesteryl ester accumulation in atherogenesis, lipoprotein formation in livers and cholesterol absorption from intestines, and is a target for treating or preventing atherosclerosis and hypercholesterolemia. During screening for microbial ACAT inhibitors, purpactins, cyclodepsipeptides and glisoprenins were previously isolated. This study discovered highly potent ACAT inhibitors termed pyripyropenes produced by Aspergillus fumigatus FO-1289. Pyripyropenes A, B, C and D were isolated from 60 liters of fermentation broth via solvent extraction, silica gel column chromatography, ODS column chromatography and HPLC, yielding pure white powders. Their molecular formulas were determined as C31H37NO10 (A) and C32H39NO10 (B-D) by HREI-MS. Structures, determined via NMR and UV spectroscopy, share a unique composition of pyridine, α-pyrone and sesquiterpene. ACAT inhibitory activity (rat liver microsomes) showed nanomolar IC50 values (53–268 nM), with pyripyropene C most potent (53 nM); the synthetic inhibitor CL 283,546 had a 1.3 μM IC50, over 20-fold less potent. Pyripyropene A inhibited cholesterol absorption in hamsters in a dose-dependent manner. Thus, pyripyropenes are highly potent ACAT inhibitors.