NHERF1/EBP50 is a PDZ-scaffold protein initially identified as an organizer and modulator of transporters and channels at the apical side of epithelia via actin-binding ezrin-moesin-radixin proteins. Presently, hepatocellular carcinoma (HCC) is one of the most deadly cancers in the world and has no effective therapeutic strategies. In the present study, we attempted to explore the role of NHERF1 in regulating liver cancer progression. The results indicated that NHERF1 was significantly expressed in liver tumor samples compared to the corresponding adjacent normal tissues. HCC patients with low NHERF1 exhibited better survival rate. Additionally, repressing NHERF1 expression markedly down-regulated the cell proliferation. G0/G1 transition was highly induced by NHERF1 knockdown, accompanied with reduced expressions of Cyclin D1 and cyclin-dependent kinase 4 (CDK4), as well as the enhanced expression of p27, phosphatase and tensin homolog (PTEN) and p53. Moreover, NHERF1 suppression significantly induced apoptosis in liver cancer cells by promoting the activation of Caspase-3 and poly (ADP-ribose) polymerase (PARP). We also observed a remarkable increase of reactive oxygen species (ROS) production in NHERF1-knockdown cells, along with c-Jun-N-terminal kinase (JNK) phosphorylation. Importantly, suppressing ROS production abolished NHERF1 knockdown-induced JNK activation. Moreover, cell cycle-regulatory proteins meditated by NHERF1 knockdown in liver cancer cells were abrogated by the pre-treatment of ROS scavenger. Further, restraining ROS generation also diminished NHERF1 knockdown-induced apoptosis. In vivo, we also found that NHERF1 knockdown markedly reduced the tumor growth. In conclusion, the results suggested that NHERF1 played an essential role in regulating liver cancer progression, and repressing NHERF1 expression exhibited significant anticancer effects via the induction of G0/G1 phase arrest, apoptosis and ROS generation.