Objective To investigate the effects of Kruppel-like factor 6 (KLF6) on the apoptotic and migration ability of HepG2 cell, and the developmental role of KLF6 on zebrafish liver. Methods Constructed plasmid with shRNA-KLF6 was transfected in HepG2 and L-02. The impacts of loss of KLF6 on HepG2 was investigated by Western bolt, apoptosis analyses, cell cycle detection and scratch experiment; KLF6 morpholino oligonucleotides was microinjected into the Tg(lfabp:eGFP) transgenic zebrafish embryos. The morphant phenotype of the liver was imaged and the protein expression of KLF6 after knockdown of KLF6 was analyzed by Immunofluorescence staining. Results The expression of KLF6 in L-02 was significantly higher than in HepG2. After knockout of KLF6, KLF6 protein expression and apoptosis were significantly reduced. In addition, the cell cycle mainly stated in S phase and the migration ability of HepG2 was enhanced. After klf6 knockdown in transgenic zebrafish larvae, the development of zebrafish liver was delayed and KLF6 expression was obviously decreased in the liver. Conclusions The reduction of KLF6 expression increased the proliferation and migration ability, and reduced the apoptosis of HepG2.Loss of KLF6 affects the development of zebrafish liver, which may open a possibility to use zebrafish as a liver cancer model and for anti-liver cancer drug screening.