Abstract: Objective: To investigate the optimal schedule of treatment with Herceptin combined with paclitaxel and5-FU in gastric cancer in vitro. Methods: The Her-2/neu overexpressing gastric cancer cell line NCI-N87 and Her-2/neu-negative gastric cancer cell line MGC803 were used. We employed MTT assay to detect the inhibitory effects and thecombined effects of Herceptin, Paclitaxel and 5-FU on gastric cancer. Flow cytometry and Western blot were used toobserve the influences on cell cycle, apoptosis and expression of Her-2, AKT, and P-AKT. Results: For MGC803 andN87 cells, the best regimen was Paclitaxel administration for 24h followed by 5 -FU administration for 48h. Thecombination of Paclitaxel and 5-FU in MGC803 cells was more effective than in N87 cells. When administered afterchemotherapeutic agents, Herceptin increased the cytotoxicity of paclitaxcel and 5-FU, decreased the percentage of cellsin S-phase and decreased the expression of Her-2 protein in N87 cells. Herceptin inhibited the expression of P-AKT inN87 cells. Conclusion: An optimized schedule combining Paclitaxel and 5-FU therapies for gastric cancer patients mayexist. Sensitivity to chemotherapy is related to Her-2/neu overexpression.