Objective To investigate the effects of Bag1 expression level on the growth of gastric cancer cells and chemo-sensitivity to 5-fluorouracil (5-FU). Methods The expression levels of Bag1 in five different gastric cancer cell lines were determined by Real-Time PCR and Western blotting. Cells with highly expressed Bag1 were selected and the basic expression level of Bag1 was down-regulated by the lentiviral shRNA. The efficiency of Bag1 silencing was confirmed by Real-Time PCR and Western blotting. The effects of the Bag1 knockdown on cell proliferation and chemo-sensitivity to 5-FU were detected by CCK-8 method. Results The expression levels of mRNA and protein of Bag1 were significantly increased in SGC7901 and KATO-Ⅲ cells. Efficient knockdown of Bag1 resulted in suppressing the proliferation of SGC7901 and KATO-Ⅲ cells and significantly enhancing the chemo-sensitivity to 5-FU. Conclusion Knockdown of Bag1 can significantly suppress the proliferation of gastric tumor cells and enhance chemo-sensitivity to 5-FU. Bag1 could be a new therapeutic target for chemotherapy of GC.