Objective To investigate the reversal effect of β-asarone on P-glycoprotein(P-gp)-mediated temozolo-mide(TMZ) resistance in glioma and its possible mechanisms. Methods TMZ-resistant U251/TMZ cell line was induced by the stepwise revulsion with TMZ. The half inhibitory concentration (IC50) and resistance index (RF) were calculated by the azole blue (MTT) assay. U251 cells were treated with β-asarone (360 μM), and the expression of P-gp/MDR1 and vimentin in the cells was detected by qRT-PCR and Western blot. Flow cytometry detected the effect of β-asarone (360 μM, 450 μM) co-administered with TMZ on apoptosis. Results The U251/TMZ cell line with stable resistance to TMZ was successfully constructed using the in vitro stepwise induction method. the P-gp expression of U251/TMZ-resistant cells was significantly increased compared with that of U251 parental cells(P<0.01). After U251 cells were treated with β-asarone, the apoptosis rate of cells in the β-asarone group was significantly increased, the invasive ability was decreased, and the protein and mRNA expression of P-gp and vimentin were significantly down-regulated compared with those in the U251 group(P< 0.01, P<0.05). Compared with the TMZ group, the protein and mRNA expression of P-gp and vimentin were significantly reduced in the TMZ+β-asarone group, and the co-administration of TMZ+β-asarone was more effective in inducing apoptosis and inhibiting invasiveness (P<0.05, P<0.01, P<0.001). Conclusion β-asarone can reverse P-gp-mediated TMZ resistance in glioma, and co-administration with TMZ may induce apoptosis and inhibit glioma cell migration and invasion by down-regulating P-gp expression.