Head and neck squamous cell carcinoma (HNSCC) ranks the eighth most common cancer worldwide. The patients often present with advanced disease, which responds poorly to chemoradiation therapy. PUMA is a BH3-only Bcl-2 family protein and a p53 target that is required for apoptosis induced by p53 and various chemotherapeutic agents. In this study, we found that PUMA induction by chemotherapeutic agents is abrogated in most HNSCC cell lines. Adenoviral gene delivery of PUMA induced apoptosis and chemosensitization more potently than did adenoviral delivery of p53 in HNSCC cells. Finally, we showed that PUMA suppressed the growth of HNSCC xenograft tumors and sensitized them to cisplatin through induction of apoptosis. Our data suggest that absence of PUMA activation in HNSCC cells contributes to chemoresistance and that gene therapy with PUMA might be an efficient substitute for p53 to enhance the responses of HNSCC cells to chemotherapy.