The transcription factor Krüppel-like factor 8 (KLF8) plays an important role in tumor development and growth, but its role in pancreatic cancer (PC) is not clear. KLF8 expression in human PC cell lines and tumor tissues was measured by quantitative real-time polymerase chain reaction and Western blot analyses. The effects of lentivirus mediated knockdown of KLF8 on proliferation and growth in Panc-1 pancreatic cancer cells were examined. KLF8 was overexpressed in 5 pancreatic cancer cell lines and in samples from patients with PC. In Panc-1 cells, KLF8 knockdown inhibited cell proliferation, tumorigenicity, and induced G2/M phase arrest. KLF8 knockdown suppressed PC tumor growth in nude mice model. Western blot analysis showed that KLF8 knockdown in Panc-1 cells down-regulated the expression of CDK1/CDC2, cyclin B1, and cyclin D1 and up-regulated the expression of p21, and p27. Overexpression of KLF8 may contribute to the progression of pancreatic cancer, and downregulation of KLF8 expression by lentivirus-delivered shRNA is a novel therapeutic approach for PC. •KLF8 was overexpressed in pancreatic cancer tissues and cell lines.•KLF8- knockdown resulted in cell proliferation suppression both in vitro and in vivo.•KLF8-knockdown influence expression of CDC2, p21, p27, resulted in G2/M arrest.•KLF8 may play an important role in PC progression.