Objective To observe the expression level of miR-141 in tumor tissues of human hepatocellular carcinoma (HCC) and determine the effect of miR-141 level on cell proliferation, invasion and migration of MHCC-97H cells by upregulation of miR-141. Methods We checked the miR-141 expression level in HCC by real-time quantitative PCR and analyzed the relationship between the expression level of miR-141 and clinical pathological indicators as well as survival rate. MHCC-97H cells were transiently transfected with miR-141 mimics which were artificially synthesized. The proliferation of MHCC-97H cells was detected by MTT assay. Transwell(TM) assay was performed to examine the invasion and migration of MHCC-97H cells. The expression of erythropoietin-producing hepatocellular receptor A2 (EphA2), which was the potential downstream target, was determined by Western blotting and immunohistochemistry. Results The expression level of miR-141 in HCC tissues was significantly lower than that in the adjacent normal tissues, and it was obviously associated with TNM stage, portal vein infiltration and Edmondson degree. Patients in the lower miR-141 group had a worse 3-year survival than those in higher miR-141 group. Overexpression of miR-141 in MHCC-97H cells significantly suppressed cell proliferation, invasion and migration, and inhibited the protein expression of EphA2. Correlation analysis showed that miR-141 level was negatively correlated with EphA2 expression level. Conclusion miR-141 is down-regulated in HCC tissues and it is negatively correlated with EphA2 expression. Its low expression is correlated with the malignant clinical pathological features. miR-141 overexpression down-regulates EphA2 expression and subsequently inhibits the proliferation, invasion and migration of HCC cells.