P-Glycoprotein (P-gp), an ATP-binding cassette transporter, plays an important role in multidrug resistance (MDR). α-Asarone and β-asarone, bioactive cis-trans isomers found in Acorus tatarinowii Schott, were tested for their potential ability to modulate the expression and function of P-gp in Caco-2 cells. MTT assays revealed that both α-asarone and β-asarone significantly enhanced the vincristine-induced cytotoxicity to cells. β-Asarone was the most potent. Flow cytometry showed that α- and β-asarone increased Rhodamine 123 (Rh123) uptake and inhibited Rh123 efflux in Caco-2 cells in a concentration-dependent manner. Furthermore, P-gp expression and P-gp mRNA in cells were decreased by exposure to α- and β-asarone. In addition, β-asarone increased the inhibition of P-gp activity in cells more than α-asarone. Thus, α- and β-asarone effectively reversed MDR by inhibiting P-gp function and expression.