Objective: To assess the anti-cancer capacity of vitamin D in head and neck squamous cell carcinoma cell line. Materials and Methods: vitamin D cytotoxic effect and cell apoptosis effect were evaluated using Prestoblue cell viability assay and Flow Cytometry. Results: Vitamin D was found to inhibit the cell growth in a dose-dependent manner in head and neck squamous cell carcinoma JHU-29 cell line at three days' treatment. However, it was not found to cause significant apoptosis at one day treatment. Conclusion: vitamin D inhibits the growth of head and neck squamous cell carcinoma in a dose-dependent manner.