The co-stimulatory molecule, CD80 (B7-1), is a ligand of CD28 and plays a key role in the induction of cell-mediated immune responses. Many tumors, including gastric cancer, have decreased expression of CD80 which leads to a failure of immune recognition. Lymph node spread is a factor of poor prognosis in gastric cancer. In this study, we transfected the CD80 gene by an adenovirus vector into a human gastric cancer cell line, OCUM-2MLN, and analyzed the effect on lymph node disease in vitro and in vivo. After transfection of CD80 in vitro, the adhesive ability of cancer cells for peripheral blood mononuclear cells and their cytotoxicity showed significant regression (p<0.01). Intratumoral injection of AdCD80 caused significantly growth of subcutaneous tumors. In vivo lymph node spread was suppressed by injection of AdCD80 into gastric tumors. Histopathologic findings revealed CD80-positive cells around the tumor. These results suggest that CD80 gene transfer into cancer cells using an adenovirus vector might be a promising approach for in vivo cancer therapy.