Objective: To investigate the expressions of miR-342-5p and its putative target gene Merlin in hepatocellular carcinoma (HCC) and the significance. Methods: The expressions of miR-342-5p and Merlin mRNA in HCC and tumor-adjacent tissues as well as in normal hepatic cell line and different HCC cell lines were determined by qRT-PCR method. HCCLM3 cells were transfected with lentiviral packaging plasmid pGCSIL-GFP-miR-342-5p or empty vector (negative control) with the untreated HCCLM3 cells as blank control, and then the invasion potential and migration activity were assessed by scratch-wound healing and Transwell migration assay, and the Merlin protein expression was measured by Western blot analysis, respectively. Using a dual-luciferase reporter assay system, the plasmids tagged with the 3’-UTR of the wild- or mutant-type Merlin gene (psiCHECK-Merlin) was co-transfected with pGCSIL-GFP-miR-342-5p or empty vectors (negative control), or transfected alone (blank control) into the HCCLM3 cells, and then the luciferase activity in each group of cells was detected. Results: Compared with tumor-adjacent tissue, the miR-342-5p expression was significantly up-regulated while Merlin mRNA expression was significantly down-regulated in HCC tissue (both P<0.05); in HCC tissues, the miR-342-5p expression was significantly higher while Merlin mRNA expression was significantly lower than that in non-vascular invasion group (P<0.05), in addition, the miR-342-5p and Merlin mRNA expression showed a negative correlation (r2=5.364, P<0.05). The miR-342-5p expression was significantly increased in all the studied HCC cell lines compared with the normal hepatic cell line, and was up-regulated with the increase of the invasion ability of the HCC cells, while the Merlin mRNA expression showed the exact opposite pattern (all P<0.05). In HCCLM3 cells transfected with pGCSIL-GFP-miR-342-5p, the invasion potential and migration activity were significantly reduced (both P<0.05) and the Merlin protein expression was elevated compared with those in blank control or negative control. In HCCLM3 cells co-transfected with psiCHECK-Merlin (wild-type) and pGCSIL-GFP-miR-342-5p, the luciferase activity was significantly decreased compared with its blank control or negative control (both P<0.05), but which showed no significant difference in HCCLM3 cells co-transfected with psiCHECK-Merlin (mutant-type) and pGCSIL-GFP-miR-342-5p compared with its blank control and negative control (P>0.05). Conclusion: Merlin is a target gene of miR-342-5p, and miR-342-5p may enhance the invasion and metastasis ability of HCC through regulating Merlin mRNA expression.