MicroRNAs (miRs) have emerged as prospective tools for human cancer therapy, including hepatocellular carcinoma (HCC) therapy. Previous studies have suggested that miR-381 functions as oncogenic or tumor-suppressive miRs in other cancer types. However, the role of miR-381 in HCC remains unknown. The present study investigated the expression and functional role of miR-381 in HCC. miR-381 expression was significantly decreased in HCC tissues and cell lines. miR-381 overexpression significantly inhibited HCC cell proliferation and colony formation, induced G0/G1 cell cycle arrest and suppressed cell invasion. Conversely, suppression of miR-381 showed the opposite effect in HCC cells. Bioinformatics analysis and dual-luciferase reporter assay results showed that miR-381 directly targeted the 3'-untranslated region of liver receptor homolog-1 (LRH-1), and quantitative polymerase chain reaction and western blot analysis results showed that miR-381 negatively modulated LRH-1 expression. Data elucidated that miR-381 directly regulated HCC cell growth and invasion, as well as the Wnt signaling pathways, by targeting LRH-1. Clinical tissue detection data revealed an inverse correlation between miR-381 and LRH-1 expression in HCC tissues, further indicating the functional significance of miR-381-LRH-1 in regulating HCC tumorigenesis. The present study indicates that miR-381 may be a novel tumor suppressor that blocks HCC growth and invasion by targeting LRH-1. The results present novel insights into understanding the molecular mechanism underlying HCC tumorigenesis and provide a future direction to the development of therapeutic interventions for HCC.