Combination of genistein (GT) and verapamil, a P-glycoprotein (P-gp) inhibitor, can increase GT absorption in situ perfusion technology in rat. To date, little information is yet available about the effect of verapamil on oral absorption of GT in vivo. In this study, a simple and reproducible HPLC–UV method was developed and validated for determination of total GT in rat plasma. Based on this, a pharmacokinetic experiment was designed to characterize biopharmaceutical properties of GT with or without coadministration of verapamil (10.0, 20.0, 30.0 mg/kg) in rats. The coadministration of verapamil (30.0 mg/kg) with GT caused a significant increase of the maximum GT plasma concentration (1.31-fold vs. GT, P < 0.05) and area under the curve (1.39-fold vs. GT, P < 0.05). Our data show that verapamil would increase intestinal absorption of GT in rat, suggesting there is some drug–nutrition interaction between verapamil and GT.