Metastasis and recurrence are major causes of death in gastric cancer patients. Because there are no obvious clinical symptoms during the early stages of metastasis, we sought to isolate highly invasive metastatic gastric cancer cells for future drug screening. We first established a mouse model to observe gastric cancer metastasis in vivo. The incidence of peritoneal metastasis of gastric cancer was much higher than liver or lymph metastasis. Peritoneal metastatic and non-metastatic NUGC-4 cells were isolated from the mouse model. Cell proliferation was measured using CCK-8 assays, while migration and invasion were investigated in Transwell assays. Proteins involved in epithelial-mesenchymal transition were detected by Western blotting. Metastatic gastric carcinoma cells were more proliferative and invasive than primary NUGC-4 cells. The supernatants of metastatic gastric carcinoma cells notably altered the morphology of HMrSV5 peritoneal mesothelial cells and promoted their epithelial-mesenchymal transition. Moreover, primary or metastatic gastric cancer cells co-cultured with HMrSV5 cells markedly increased cancer cell proliferation and invasiveness. Moreover, peritoneal metastatic gastric carcinoma cells in the presence of HMrSV5 cells exhibited most malignant behaviors. Thus, peritoneal metastatic gastric carcinoma cells exhibited high capacities for proliferation and invasion, and could be used as a new drug screening tool for the treatment of advanced gastric cancer and peritoneal metastatic gastric cancer.