Objective: To compare the sensitivity to chemotherapeutic agents between the tumor cells from primary and metastatic site in nude mice with orthotopic implantation of human gastric cancer. Methods: Orthotopic xenograft models of human gastric cancer were established in nude mice by intra-gastric wall inoculation of the fragments of tumor mass derived from SGC7901 cells which were subcutaneously grown in the donor mice. After metastases formation, the disaggregated tumor cells from primary site and liver metastases were collected, and the in vitro sensitivities of the cells to seven chemotherapy drugs that included fluorouracil (5-FU), cisplatin (CDDP), oxaliplatin (L-OHP), epirubicin (eADM), mitomycin (MMC), vincristine (VCR) and methotrexate (MTX) were determined by sulforhodamine B (SRB) assay. Results: Orthopotic mouse models of stomach cancer were established successfully. The rate of primary tumor formation following orthopotic implantation was 100%, and the liver metastasis rate was 75%. Among the seven drugs, the inhibition rates of L-OHP and VCR for the tumor cells from primary site were higher than those from metastases, while the inhibition rates of eADM and MMC for the tumor cells from metastases were higher than those from primary site (all P<0.05); the inhibition rates of 5-FU, L-OHP, MTX for the tumor cells between primary and metastatic sites had a positive correlation (r=0.5203, 0.4424 and 0.3851, all P<0.05). Conclusion: Chemotherapy sensitivity varies between the tumor cells from primary site and hepatic metastasis in nude mice with orthotopic implantation of stomach cancer, so the treatment for liver metastasis that is based on the drug sensitivity testing results from the primary tumor may not be accurate.