Kruppel-like factor 8 (KLF8) is a transcription factor which has been identified to play a critical role in oncogenic transformation, epithelial-mesenchymal transition and invasion. Higher expression level of KLF8 has been observed in ovarian, renal and breast cancer cells. This study focused on investigating the knockdown effects of KLF8 through lentivirus mediated targeted disruption of KLF8 in gastric cancer cell lines. The expression level of KLF8 is much higher in gastric cancer cells than that in normal cell via Western blot analysis. The decreased expression level of KLF8 after repression was confirmed by real-time PCR and Western blot in SGC-7901, a gastric cancer cell line. The effects of KLF8 deletion on cell proliferation and cell cycle were analyzed by MTT assay and flow cytometry, respectively. Finally, the effects of KLF8 deletion on cell apoptosis and invasion of gastric cancer cells were analyzed by Annexin staining and transwell assay, respectively. It was observed that knockdown of KLF8 reduced the cellular proliferation of SGC-7901 gastric cancer cells, a phenotype at least partially due to cell cycle arrest at G1 phase and increased apoptosis. Furthermore, the inhibition of KLF8 reduces the invasion rates of the cancer cells. Therefore, KLF8 is necessary for cell survival and invasion in gastric cancer cells. The absence of KLF8 may lead to cancer cell death. These results demonstrated that the lentivirus mediated targeted disruption of KLF8 would be an promising therapeutic method for treatment of gastric cancer.