We performed the study to investigate whether adenovirus-mediated retinoblastoma 94 (RB94) gene transfer could enhance radiation treatment of esophageal squamous cell carcinoma (ESCC) in vitro and in vivo. ESCC cells (Kyse150 cell line) were cultivated in vitro and tumors originated from the cell line were propagated as xenografts in nude mice. Treatment with Ad-RB94 and/or ionizing radiation (IR) was carried out both in vitro and in vivo with Ad-LacZ control vector and blank control. Cell viability, cell cycle distribution, cell apoptosis, tumor growth and transfected gene expression were evaluated and tumor degeneration was analyzed. The data of quantification real-time PCR assays and immunohistochemistry staining using RB antibody indicated that RB94 was efficiently transfected into Kyse150 cells. In vitro, data of cell growth assay indicated that treatment with Ad-RB94 improved radiation treatment of Kyse150 cells. Tumor xenograft studies, pathological analysis of H.E. staining and Ki67 staining suggested transfecting RB94 enhanced tumor regression induced by radiation treatment in vivo. In addition, data of Annexin V, TUNEL and cell cycle distribution assays proposed combination treatment effectively induced cell apoptosis and cell cycle arresting in G2/M phase. In conclusion, transferring RB94 gene by the adenoviral vector enhances radiation treatment of ESCC.