Background Enalapril is an angiotensin-converting enzyme (ACE) inhibitor approved for the treatment of mild to severe hypertension and congestive heart failure. There is evidence that enalapril may be an organic anion-transporting polypeptide 1B1 (OATP1B1) substrate, suggesting that genetic polymorphisms of the OATP1B1 gene may play a role in causing the interindividual pharmacokinetic differences of this drug. Objective The purpose of this study was to investigate the functional significance of the OATP1B1 genetic polymorphism on the pharmacokinetics of enalapril and its active metabolite enalaprilat in healthy Chinese adult male participants. Methods This was a single-center, open-label, single- and multiple-dose study conducted in healthy Chinese male participants. Each participant received a single oral dose of 10 mg enalapril under fasting conditions, followed by enalapril 10 mg/d for 7 days. In the single-dose phase, sequential blood samples were collected from 0 to 24 hours after drug administration. In the multiple-dose phase, samples were obtained before drug administration on days 4, 5, 6, and 7; on day 7, samples were collected from 0 to 72 hours after drug administration. An HPLC-MS/MS method was used to determine plasma concentrations of enalapril and enalaprilat. A polymerase chain reaction technique was used for genotyping of 2 single nucleotide polymorphisms (SNPs) of the OATP1B1 gene: T521C and A388G. The pharmacokinetic parameters of enalapril and enalaprilat were then compared according to genotype groups, using 1-way ANOVA, except for T max in which the Mann-Whitney test or Kruskal-Wallis test was used. Results The study included 32 healthy Han Chinese male participants (age range, 18–28 years; weight range, 50.0–80.0 kg; height range,159–182.0 cm). Twenty-six were OATP1B1*15 noncarriers (homozygous for 521TT), the others were *15 carriers with at least one 521 T>C mutant allele. After single and multiple oral doses of 10 mg enalapril, plasma concentrations of enalapril in *15 noncarriers were lower than that in *15 carriers, with significant difference in area under the curve at steady state (AUC ss ) between *15 noncarriers and *15 carriers ( P = 0.048) in the multiple-dose phase. There were no significant differences in enalapril's AUC 0–24 , C max , or the ratio of the AUC 0–24h in the single-dose study to the AUC ss (R ac ) between the *15 carriers and noncarriers. In contrast to enalapril, the mean AUC 0–24h and C max of enalaprilat in *15 noncarriers was significantly higher than those in *15 carriers ( P = 0.040 and P = 0.027, respectively) in the single-dose phase. There were no significant differences in enalaprilat's AUC ss or C maxss between the 2 groups in the multiple-dose phase. For the 3 groups classified according to the effect of A388G variant in all subjects homozygous for 521T (TT), *1a/*1a, *1a/*1b, and *1b/*1b, no significant difference was found in AUC 0–24h , C max , and T max of enalapril and enalaprilat. Conclusions In this small population of healthy Chinese men, the OATP1B1*15 allele and T521C variant appeared to be an important determinant of the pharmacokinetics of enalapril. There were significant differences between the *15 carriers and noncarriers in enalapril's AUC ss and enalaprilat's AUC 0–24h , C max , and R ac . However, there were no significant differences in enalapril's AUC 0–24 , C max , or enalaprilat's AUC ss , C maxss between the *15 carriers and noncarriers.