Aims: The cholesterol-lowering drug simvastatin is a substrate for P-glycoprotein (P-gp). P-gp, encoded by ABCB1, is an efflux transporter and genetic variation in ABCB1 is associated with drug levels and response. We examined the Rotterdam Study, which is a population-based cohort study of people aged 55 years and older, to see whether the C1236T, G2677T/A and C3435T polymorphisms and haplotypes in the ABCB1 gene are associated with the total cholesterol and low-density lipoprotein cholesterol-lowering effect of simvastatin. Materials & methods: We identified 85 incident simvastatin users, for whom a cholesterol measurement both before and after the start of simvastatin therapy was available. Associations between the ABCB1 gene variants and reductions in cholesterol levels were analyzed. In our analysis we stratified for gender, because the level of P-gp expression in the liver is higher in men than in women. Results: The three ABCB1 polymorphisms were associated with total cholesterol reduction in the whole population. In men, both the 1236/2677/3435 TTT haplotype and the CGT haplotype were associated with larger reductions in total cholesterol (TTT: -0.40 mmol/l, 95% Cl: -0.63 to -0.17; CGT: -0.44 mmol/l, 95% Cl: -0.77 to -0.11) and low-density lipoprotein cholesterol levels (TTT: -0.51 mmol/l, 95% Cl: -0.81 to -0.22; CGT: -0.53 mmol/l, 95% Cl: -0.92 to -0.15) than the reference CGC haplotype. In women, genetic variation in the ABCB1 gene was not associated with total and low-density lipoprotein cholesterol levels. Conclusion: Male simvastatin users with the ABCB1 1236/2677/3435 TTT and CGT haplotype have larger reductions in total cholesterol and low-density lipoprotein cholesterol compared with the wild-type CGC haplotype. For women, no associations were found.