IFN-γ plays an indirect anti-cancer role through the immune system but may have direct negative effects on cancer cells. It regulates the viability of gastric cancer cells, so we examined whether it affects their proliferation and how that might be brought about. We exposed AGS, HGC-27 and GES-1 gastric cancer cell lines to IFN-γ and found significantly reduced colony formation ability. Flow cytometry revealed no effect of IFN-γ on apoptosis of cell lines and no effect on cell aging as assessed by β-gal staining. Microarray assay revealed that IFN-γ changed the mRNA expression of genes related to the cell cycle and cell proliferation and migration, as well as chemokines and chemokine receptors, and immunity-related genes. Finally, flow cytometry revealed that IFN-γ arrested the cells in the G1/S phase. IFN-γ may slow proliferation of some gastric cancer cells by affecting the cell cycle to play a negative role in the development of gastric cancer.