Although Epstein-Barr virus (EBV) is associated with 6-16% of the gastric carcinoma (GC) cases, the effect of EBV infection on the tumorigenesis process and the responsiveness to chemotherapy remain unclear. We compared chemosensitivity of the EBV-positive GC (AGS-EBV) and EBV-negative GC (AGS) cells to 5-fluorouracil (5-FU). Although 5-FU inhibited the growth of both cell lines in a dose- and time-dependent manner, the sensitivity of EBV-positive GC cells to 5-FU was lower than that of EBV-negative GC cells. The cleavage of PARP and caspase-3 was also lower in AGS-EBV cells than in AGS cells following 5-FU treatment. Both the level of Bcl-2 expression and the ratio of Bcl-2/Bax were higher in AGS-EBV than in AGS cells not only at basal state but also following 5-FU treatment. Moreover, p53 and p21 expression was enhanced further by 5-FU in AGS than in AGS-EBV cells. Immunofluorescence assay and Western blot showed that 5-FU induced the expression of EBV-lytic genes including BZLF1, BRLF1, BMRF1 and BHRF1. Our results suggest that latent and lytic EBV infection contributes to the chemoresistance to 5-FU in gastric carcinoma by modulating apoptosis related cellular genes.