Lemur tyrosine kinase 2 (LMTK2) was recently identified as a novel cancer-related gene in several human cancers. However, little is known of its function in hepatocellular carcinoma (HCC). Here we aim to investigate the expression pattern, biological function, and regulatory mechanism of LMTK2 in HCC. We found that LMTK2 was highly expressed in HCC tissues, and patients with high expression of LMTK2 in tumor tissues had shorter survival times. LMTK2 expression was also elevated in HCC cell lines, and LMTK2 silencing markedly repressed the proliferation and invasion of HCC cells. By contrast, LMTK2 overexpression exerted promotion effects on HCC cell proliferation and invasion. Our results demonstrate that LMTK2 silencing decreases the phosphorylation of glycogen synthase kinase-3 beta (GSK-3 beta and the expression of an active beta-catenin protein, leading to inhibition of Wnt/beta-catenin signaling. Notably, GSK-3 beta inhibition significantly reversed the LMTK2 silencing-mediated antitumor effect on proliferation, invasion, and Wnt/beta-catenin signaling in HCC cells. LMTK2 silencing retarded the tumor growth of HCC cells in an in vivo xenograft tumor model, associated with downregulation of Wnt/beta-catenin signaling. In conclusion, our findings suggest that silencing of LMTK2 suppresses the proliferation and invasion of HCC cells through the inhibition of Wnt/beta-catenin signaling, via GSK-3 beta, highlighting the importance of LMTK2/GSK-3 beta/Wnt/beta-catenin signaling in HCC progression. (C) 2019 Elsevier Inc. All rights reserved.