A truncated retinoblastoma (RB) protein of approximately 94 kDa (RB94), lacking the NH2 -terminal 112 amino acid residues of the full-length RB, has been found to have great efficacy in tumor suppression. This study investigated the role of adenovirus-mediated RB94 (Ad-RB94) gene therapy for human head and neck squamous cell carcinoma (HNSCC) and explored the cellular and molecular mechanism of tumor inhibition after Ad-RB94 gene transfer. Randomized controlled studies in vitro and in vivo were performed to assess antitumor responses of Ad-RB94 gene transfer against human HNSCC. Human HNSCC cell lines, JHU006 and JHU012, were used in this study. Tumors originated from the HNSCC cell lines were propagated as xenografts in nude mice. Ad-RB94 gene transfer was performed both in vitro and in vivo with replication-defective virus (DL312) and no treatment as controls. Transgene expression, cell viability, and tumor growth were evaluated in transfected cells and tumor implants. To determine the mechanism behind the observed antitumor action, cell cycle analysis was performed, and telomerase activity was examined. Tumors were evaluated for RB94-induced apoptosis. Transgene expression of RB94 was detected by Western blot analysis, real-time quantification reverse transcription-PCR, and immunohistochemistry. RB94 expression led to flattening of cell growth curves and caused tumor regression. Animals treated with Ad-RB94 were seen to have a significant reduction in tumor size when compared with DL312 (P = 0.02, both cell lines) and to no treatment groups (P = 0.01, both cell lines). Cell cycle arrest in the G(2)-M phase and increased levels of apoptosis occurred in tumor cells treated with Ad-RB94. In addition, telomerase activity decreased significantly and specifically after Ad-RB94 treatment. This study demonstrates that Ad-RB94 gene transfer effectively inhibits HNSCC tumor cell growth in vitro and in vivo. The unique property of Ad-RB94 gene transfer to arrest HNSCC tumor cells in the G2-M phase of the cell cycle makes it a good candidate for adjuvant therapy with radiation or chemotherapy, as tumor cells are most sensitive to radiation or cytotoxic drug in this cell cycle phase.