To determine the effect of genistein on cytochrome P450 3A (CYP3A) and P-glycoprotein (P-gp) function using the probe substrates midazolam and talinolol, respectively. Eighteen healthy adult male participants were enrolled in a two-phase randomized crossover design. In each phase, the participants received placebo or genistein for 14 days. On the 15th day, midazolam and talinolol were administered and blood samples were obtained. Midazolam and talinolol pharmacokinetic parameter values were calculated and compared before and after genistein administration. Co-administration of genistein decreased the area under the concentration-time curve from 0 to 36 h (AUC 0-36) (143.65 ± 55.40 ng h/mL versus 126.10 ± 40.14 ng h/mL, p < 0.05), and the area under the concentration-time curve from zero to infinity (AUC 0-∞) (209.18 ± 56.61 ng h/mL versus 180.59 ± 43.03 ng h/mL, p < 0.05), and also maximum concentration (Cmax) of midazolam (48.86 ± 20.21 ng/mL versus 36.25 ± 14.35 ng/mL p < 0.05). Similarly, AUC 0-36 (2490.282 ± 668.79 ng h/mL versus 2114.46 ± 861.11 ng h/mL, p < 0.05), AUC 0-∞ (2980.45 ± 921.09 ng h/mL versus 2626.92 ± 1003.78 ng h/mL, p < 0.05) and Cmax of talinolol (326.58 ± 197.67 ng/mL versus 293.42 ± 127.19 ng/mL, p < 0.05) were reduced by genistein co-administration. The oral clearance of midazolam (1.68 ± 0.85 h-1 versus 3.98 ± 0.59 h-1, p < 0.05) and talinolol (3.34 ± 1.24 h-1 versus 3.79 ± 1.55 h-1, p<0.05) were increased by genistien significantly. Administration of genistein can result in a modest induction of CYP3A and possibly P-gp activity in healthy volunteers.