To explore the protective effect of KLF4 against cytotoxicity induced by cisplatin and its possible mechanisms. The expression levels of KLF4 were detected by RT-PCR and western blot in cancer stem-like cells derived from hepatocarcinoma (T3A-A3) and the hepatocarcinoma cell line HepG2. KLF4 was knocked down in T3A-A3 by infection of pLVTHM-shKLF4 lentivirus and ectopic expressed in HepG2 by infection of pWPTS-KLF4 lentivirus. The MTT assay was carried out to determine the impact of KLF4 on cell survival in response to cisplatin. Cisplatin-induced DNA damage was measured by TUNEL staining. Glutathione content was measured by enzymatic assay. Buthionine sulfoximine was used to deplete the content of glutathione. The expression of gamma-glutamylcysteine synthetase was analyzed by RT-PCR in HepG2 cells ectopic expressed KLF4. With a higher level of KLF4, T3A-A3 cells were found to be more resistant to cisplatin than HepG2 cells. KLF4 knockdown was found to reduce cisplatin resistance in T3A-A3 cells. Ectopic expression of KLF4 in HepG2 cells was found to be associated with heightened resistance to DNA damage after exposure to cisplatin. Furthermore, the content of glutathione was found to be higher in T3A-A3 cells than in HepG2 cells. A nearly twofold increase in the cellular level of glutathione was identified in HepG2 cells with ectopic expression of KLF4. This was accompanied by heightened resistance to cisplatin. KLF4-mediated resistance to cisplatin in HepG2 cells was found to be completely abrogated by treatment with buthionine sulfoximine, an inhibitor of glutathione synthesis, which did not affect the expression of KLF4. Moreover, the mRNA expression of gamma-glutamylcysteine synthetase, a rate-limiting enzyme of glutathione synthesis was up-regulated by KLF. We conclude that KLF4 regulates the cellular sensitivity to cisplatin in hepatocarcinoma stem-like cells and hepatocarcinoma cells by elevating intracellular glutathione levels.