Hepatocellular carcinoma (HCC) results in large amounts of deaths each year worldwide. To develop more effective treatments for HCC, it is very necessary to define the molecular mechanisms in hepatocarcinogenesis. Mixed lineage kinase (MLK)-4 is a member of the MLK family of mitogen-activated protein kinase kinase kinases, and modulates different cellular responses. However, its role in the meditation of HCC progression remains unclear. In the study, we found that MLK4 was over-expressed in tumor samples of HCC patients. High MLK4 expression was significantly associated with shorter overall survival in HCC. Knockdown of MLK4 inhibited HCC cell proliferation and metastasis, which was partly through reducing matrix metalloproteinase (MMP)-13, MMP2, enhancer of zeste homolog 2 (EZH2) and Vimentin expressions. Apoptosis was significantly induced by MLK4 knockdown in HCC cells via decreasing Bcl-2 and increasing cleaved poly (ADP-ribose) polymerase (PARP), Caspase-7 and -3 expression levels. In addition, MLK4 silence led to a significant reactive oxygen species (ROS) production in liver cancer cells, accompanied with elevated expression of phosphorylated p38, c-Jun N-terminal kinase (JNK) and ERK1/2. Notably, reducing ROS generation and blocking MAPKs (p38/JNK/ERK1/2) signaling markedly abrogated MLK4 knockdown-induced apoptosis in HCC cells. Moreover, MLK4 silence-prevented metastasis was also rescued by scavenging ROS generation and repressing MAPKs pathway. In vivo, injection of MLK4 siRNA markedly inhibited liver tumor growth in xenograft models, and MLK4 knockdown reduced HCC lung metastasis. Together, our study indicated the essential function of MLK4 in HCC progression, providing crucial therapeutic hypothesis for the prevention of hepatocellular carcinoma.