5-Fluorouracil (5-FU) plays an important role in the chemotherapy of advanced gastric cancer. However, genetic factors that affect therapeutic efficacy of 5-FU warrant further investigation. In the present study, using stable transfection of the ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) gene, we explored the genetic influences on 5-FU-induced apoptosis of human gastric cancer cells. Stable overexpression of the EBP50 gene was determined by reverse transcription polymerase chain reaction (RT-PCR) assay and western blot analysis. After treatment with 5-FU, cell growth activities in vitro were investigated by MTT assay. Cell apoptosis was evaluated by Hoechst 33258 staining and flow cytometry of Annexin V-FITC/PI staining. Compared with the BGC823 or BGC823/neo cells, EBP50 mRNA and protein levels in the BGC823/EBP50 cells (EBP50-transfected BGC823 cells) were markedly higher. Chemosensitivity and apoptosis rates of the BGC823/EBP50 cells were higher compared to the BGC823 and BGC823/neo cells following treatment with 5-FU. Stable overexpression of extrinsic EBP50 distinctly increases the 5-FU-induced apoptosis of gastric cancer cells, and is a novel strategy by which to improve the chemosensitivity of gastric cancer to 5-FU.