Objective:To investigate if in vitro chemotherapy can induce the EMT progress in gastric cancer (GC) cells. Method:The GC cell line, SGC7901, was treated using 5-Fu at a concentration of 30 μg/mL. The residual cells after four cycles of 5-Fu therapy were named as SGC7901/Fu. The morphological changes and malignant biological features, including the invasiveness and clone formation ability and the characteristics of cancer stem cell and biomarkers of EMT between SGC7901 and SGC7901/Fu, were compared. Results:The SGC7901/Fu cells displayed a mesenchymal appearance, decreased the expression of epithelial markers, and increased the expression of mesenchymal markers. The 50% inhibitory concentrations in the SGC7901/Fu and SGC7901 cells were (43.8 ± 7.2) and (64.6 ± 5.5)μg/mL, respectively. The number of cells that migrated through the basement-membrane of the Transwell chamber was 51.4 ± 8.7 and 93.2 ± 9.5, respectively. The rate of clone formation was 5.2%± 1.0%and 13.2%± 2.2%, respectively. The portions of the CD44+/CD24-cells were 4.13%±0.81%and 7.97%±0.50%, respectively. All differences were statistically significant (P<0.05). Conclusion:The residual GC cells underwent EMT progress after 5-Fu treatment, with increased chemoresistance and ability of invasiveness and acquired the property of cancer stem cells.