Hepatocellular carcinoma (HCC) is a major public health concern because of the absence of early diagnosis and effective treatments. Efficient diagnosis modalities and therapies to treat HCC are needed. Kruppel-like factor (KLF) family members, such as KLF6, are involved in cell proliferation and differentiation. KLF6 is inactivated in solid tumors, which may contribute to pathogenesis. However, KLF6 status in HCC is controversial. Thus, we undertook the characterization of KLF6 expression and function in HCC and HCC-derived cell lines. We found that HCC, HepG2 and HuH7 cells expressed KLF6 messenger ribonucleic acid and protein. Next, using RNA interference, we demonstrated that inhibiting KLF6 expression in vitro strongly impaired cell proliferation-induced G1-phase arrest, inhibited cyclin-dependent kinase 4 and cyclin D1 expression, and subsequent retinoblastoma phosphorylation. Finally, KLF6 silencing caused p53 upregulation and inhibited Bcl-xL expression, to induce cell death by apoptosis. Taken together, these data demonstrated that KLF6 is essential for HCC-derived cells to evade apoptosis.