Abstract: Objective To investigate the effect of tetrandrine (TET) on intestinal absorption by regulating the function of P-glycoprotein (P-gp). Methods The permeability volume and coefficient of a P-glycoprotein substrate, rhodamine123 (Rho123) on the intestinal absorption and emptying directions were determined by an in vitro diffusion chamber system. The effects of TET on Rho123 and 5(6)-carboxy fluorescein (CF) on in vitro permeability of rate intestinal mucosa were detected. Results TET could stimulate the absorption of Rho123 and inhibit its emptying, which was not related to the paracellular pathway. Conclusions TET may inhibit the activity of P-gp in the intestine. TET can promote the absorption of oral tumor drugs and inhibit multidrug resistance, so as to reduce toxicity and enhance effectiveness.