Abstract: Objective: To investigate the effect of berberine on the cell viability and apoptosis in human oral squamous cell carcinoma cell line HN-4, and explore the possible molecular mechanisms. Methods: The cultured HN-4 cells were treated with different concentrations of berberine for 24 or 48 h; then detected by MTS method to measure its viability. The apoptosis rate was quantified by using Annexin V-FITC apoptoticdetection kit. The mitochondrial membrane potential changes were detected with Rhodamine 123 dye by flow cytometric assay. The expression of Bax, Bcl-2, Cytochrome C, cleaved-Caspase 3 and cleaved-Caspase 9 was detected by Western Blot. Results: Berberine significantly inhibited the viability of HN-4 cells in a dose- -dependent manner. Berberine significantly increased the apoptosis rate and induced theloss of mitochondrial membrane potential on HN-4 cells.Berberine significantly upregulated the expression of pro-apoptotic proteins including Bax, Cytochrome C, cleaved-Caspase 3 and cleaved-Caspase 9, as well as upregulated the expression of anti-apoptotic Bcl-2 protein. Conclusions: Berberine significantlyinhibited the viability and induced apoptosis of HN-4 cells through activating mitochondrial-mediated pathway.