Abstract: This study is designed to investigate the effect of triptolide on the function and expression of P-glycoprotein (P-gp) in HNE1 nasopharyngeal cancer cells. MTT assay was used to test cell viability. Intracellular doxorubicin content was evaluated with flow cytometry. Rhodamine 123 (Rh) was used to detect the excretion function of P-gp. The expression of P-gp was analyzed by Western blot. ATP levels were evaluated. JC-1 staining was used to determining mitochondrial membrane potential (MMP). Triptolide, doxorubicin and the combination treatment all had the inhibitory effect to HNE1 cells, and the combination treatment had the best effect. Triptolide increased intracellular concentration of doxorubicin and Rh (P < 0.05 or P < 0.01), inhibited the excretion function of P-gp. The expression of P-gp was reduced greatly in the middle and high dose group of triptolide. The ATP levels were decreased significantly (P < 0.05). JC-1 staining showed that triptolide mediated the down-regulation of MMP in HNE1 cells. Triptolide could increase intracellular drug content and enhance cytotoxicity of chemotherapeutics by inhibition of the expression and the excretion function of P-gp.