Human adenovirus (HAdV) vectors are intensely investigated for virotherapy of a wide variety of human cancers. Here, we have evaluated the effect of two apoptogenic HAdV5 vectors in an immunocompetent Syrian hamster animal model of head and neck cancer. We established two cell lines of hamster cheek pouch squamous cell carcinomas, induced by treatment with 9,10-dimethyl-1,2-benzanthracene. These cell lines, when infected with HAdV5 mutants lp11w and lp11w/Δ55 K (which are defective in the expression of either E1B-19 K alone or both E1B-19 K and E1B-55 K proteins) exhibited enhanced apoptotic and cytotoxic responses. The cheek pouch tumor cells transplanted either subcutaneously at the flanks or in the cheek pouches of hamsters readily formed tumors. Intratumoral administration of HAdV5-E1B mutants efficiently suppressed the growth of tumors at both sites. Histological examination of orthotopic tumors revealed reduced vascularity and the expression of the viral fiber antigen in virus-administered cheek pouch tumors. These tumors also exhibited increased caspase-3 levels, suggesting that virus-induced apoptosis may contribute to tumor growth suppression. Our results suggest that the apoptogenic HAdV5 vectors may have utility for the treatment of human head and neck cancers.