3, 5-Hydroxy -6, 7, 3′, 4′-tetramethoxyflavone (HTMF) isolated from Laggera pterodonta is known to have an antiproliferative effects in vitro on human cancer. However, the exact mechanisms retain unclear. HTMF was investigated for its antiproliferative effects on human nasopharygeal carcinoma CNE cells. 3-(4, 5-Dimethylthiazol-2-yl)- 2, 5-diphenyl tetrazolium bromide (MTT) assay was used to deserve the inhibitory effect of HTMF. The changes of the cell and nuclear morphological characteristics were observed under the inverted and fluorescence microscope. The cell apoptosis was displayed by Hoechst 33258 staining and flow cytometry (FCM). The expression of Caspase3 and Caspase9 was detected by Western blotting. The mitochondrial membrane potential were analyzed by FCM and laser confocal microscope with Jc-1 fluorescence staining. MTT assay results show that HTMF significantly inhibited the growth of CNE cells in dose and time dependent manners. The IC₅₀ values of HTMF were 69.02, 28.31 and 3.95 mg/L at 24, 48 and 72 h treatments, respectively. The apoptosis percentage in CNE cells is significantly increased compaired with control group. HTMF with 0, 5.0, 10.0, 20.0 and 40.0 mg/L at 48 h and 40.0 mg/L at 0, 6, 12, 24 and 48 h treatments, respectively increased the expression of Caspase3 and Caspase9 and degraded mitochondrial membrane potential in dose and time dependent manners. The mechanistic investigation revealed that HTMF has high inhibitory effects on the proliferation of CNE cells and induced the apoptosis of CNE cells by the decrease in mitochondrial membrane potential and increasing the expression of Caspase3 and Caspase9.