Larynx squamous cell carcinoma represents one of the most common head and neck cancers in the world. Herbal drugs are popularly emerging as complementary and alternative therapies in cancer because of their cost effectiveness and minimal side effects. The present study was undertaken to explore the anti-tumor potential of berberine, an isoquinolone present in the extract of Tinospora cordifolia in HEP2 human laryngeal cancer cell line. Besides, it was aimed to investigate whether berberine could enhance the anti-cancer effect of 5-fluorouracil and cisplatin in HEP2. Our data seem to support a role for berberine in decreasing the expression of genes usually seen overexpressed in larynx squamous cell carcinoma and involved in pathways such as those of cell cycle and regulation, differentiation, and epithelial-mesenchymal transition. Moreover, a down regulation of these genes caused by cisplatin or 5-fluorouracil, treatment of election in laryngeal cancers was enhanced by a 4h pre-treatment with berberine.