Increased expression of sialyl Lewis x/a carbohydrates, ligands for E-selectin, correlates with clinically advanced stages and metastasis of gastric and colon cancers. In contrast, Sd a carbohydrate is abundantly detected in the normal gastrointestinal mucosa but dramatically reduced or lost in cancer tissues. A glycosyltransferase, β1,4N-acetylgalactosaminyltransferase 2 (B4GALNT2) that catalyzes Sd a carbohydrate synthesis, is silenced in cancer. In the present study, we aimed at reducing the expression of sialyl Lewis x/a of cancer cells in vivo by forced expression of B4GALNT2 and Sd a, thereby preventing dissemination/metastasis, especially metastasis triggered by surgical maneuvers. We used a fiber-modified adenovirus (Ad) vector that contained a chimeric construct with a serotype 5 shaft and a serotype 3 knob. Using this Ad5/3 vector, we successfully introduced the B4GALNT2 gene into a human gastric cancer cell line KATO III in vitro and confirmed replacement of sialyl Lewis x to Sd a with a decrease in E-selectin-dependent adhesion. Administration of Ad5/3-B4GALNT2 vectors into the peritoneal cavity of mice after inoculation of KATO III cells with laparotomy significantly reduced the incidence of metastasis. Our results indicate that the transfer of a single gene encoding B4GALNT2 modified carbohydrate chains of cancer cells in vivo and decreased tumor dissemination and metastasis.