Abstract Background It has been reported that pomegranate juice significantly increased the AUC of orally administered carbamazepine in rats, which suggests that pomegranate may inhibit the cytochrome P450 3A (CYP3A)–mediated carbamazepine metabolism. Objective The aim of the present study was to clarify the effect of repeated consumption of pomegranate juice on CYP3A activity by assessing the pharmacokinetics of midazolam, a typical CYP3A probe drug, and its metabolites in healthy volunteers. Methods An open-label, randomized, single-center, 2-period crossover study was conducted on healthy Japanese volunteers. Each subject received 200 mL of pomegranate juice twice daily for 2 weeks. On day 14, they were administered 15 μg/kg midazolam orally with either pomegranate juice or water. Plasma concentrations and urinary excretions of midazolam, 1′-hydroxymidazolam, and 4-hydroxymidazolam were determined up to 24 hours using LC/MS/MS and analyzed by a noncompartmental method. Results Sixteen subjects (11 men and 5 women) were enrolled and completed the study. The mean (SD) age was 24.1 (4.8) years (range 22–40), mean body weight was 62.9 (8.8) kg (range 45.6–79.9). Differences in the mean AUC0–∞ were 12.7 (4.4) and 14.2 (6.6) ng/mL/h in pomegranate juice and control groups, respectively (geometric mean ratio: 1.02 95% CI, 0.95–1.09; P = 0.40). Differences in Cmax for midazolam did not reach the level of statistical significance (5.1 1.7 vs 5.0 2.0 ng/mL, geometric mean ratio: 0.95 95% CI, 0.79–1.11; P = 0.68). Excretions of 1′-hydroxymidazolam ( P = 0.34) and 4-hydroxymidazolam ( P = 0.32) were not significantly altered by ingestion of pomegranate juice. Conclusion In this small Japanese adult volunteer population receiving single subtherapeutic doses of midazolam, 2 weeks' consumption of pomegranate juice did not significantly alter the pharmacokinetic profile of midazolam compared with that of the control. Protocol identifier: UMIN000004459.