Acyl-CoA: cholesterol acyltransferase-2 (ACAT2), an intracellular cholesterol esterification enzyme found only in the intestine and liver, has been demonstrated to be associated with hypercholesterolemia and atherosclerosis in mice. To explore the possible impact of ACAT2 gene variants on CAD susceptibility and plasma lipid levels, three polymorphisms, 41A>G (Glu>Gly), 734C>T (Thr>Ile), and IVS4-57_58 ins48 bp (D/I), were genotyped in 809 CAD patients (CAD+) and 1,304 controls (CAD-) from three distinct Singaporean ethnic groups (1,228 Chinese, 367 Malays and 518 Indians). The 734T allele frequency was significantly lower in CAD+ (0.20) than CAD- (0.26) in Chinese (P=0.003) and I allele of D/I was significantly higher in CAD+ (0.17) than CAD- (0.10) in Indians (P=0.011). The 41G allele was significantly more frequent among normolipidemic (0.19) than dyslipidemic (0.13) individuals in Chinese (P=0.008). In normolipidemic females, 734C>T was associated with apoA1, apoB and lipoprotein (a) in Indians, and with apoA1 in Malays, whereas 41A>G is associated with total cholesterol in Indians. The 734C>T polymorphism was in almost complete linkage disequilibrium (LD) with the IVS4-57_58 ins48 bp and in very strong LD with 41A>G in all the three ethnic groups. In the normolipidemic females, the AG/CT had much higher apoB than AA/CC in Indians. We found that the three ACAT2 polymorphisms studied are associated with CAD risk and plasma lipid levels but their effects are not consistent across genders and ethnic groups.