Abstract: To explore the effects of Zhimu extract on the absorption and pharmacokinetic process of puerarin, thus to elucidate the scientific connotation of the compatibility application of Gegen-Zhimu herb pair from the perspective of P-glycoprotein (P-gp) regulation, a rapid and sensitive quantification method for puerarin in biological samples was established based on HPLC-UV technology. Combined with the rat single-pass intestinal perfusion model and pharmacokinetic experiments, with the classic P-gp inhibitor verapamil as control, the effects of Zhimu extract on the absorption and metabolism process of puerarin were investigated. The established HPLC method for puerarin in intestine perfusate and plasma samples met the requirements for quantitative analysis of biological samples. The K_a and P_app values of puerarin in the duodenal segment were significantly higher than those in the jejunal and ileal segments (P < 0.000 1). Co-administration with Zhimu extract or verapamil significantly increased the K_a and P_app values of puerarin in the duodenal segment (P < 0.000 1). Co-administration with high-dose Zhimu extract or verapamil significantly increased the C_max of puerarin in rats to (1.017 ± 0.25) µg·mL^-1 and (1.827 ± 0.87) µg·mL^-1 (P < 0.05), respectively, and AUC_0-∞ also significantly increased (P < 0.05), reaching 1.37-fold and 2.25-fold that of the puerarin group, respectively. Puerarin is best absorbed in the duodenal segment; by inhibiting the efflux of P-gp, Zhimu extract can promote the absorption of puerarin in the duodenal segment, increase its plasma concentration, and effectively improve the oral bioavailability of puerarin, which suggests the mechanism underlying the compatibility of Gegen-Zhimu herb pair. This experiment was approved by the Animal Ethics Committee of Naval Medical University (approval No.: NMUARE-21077).