Hepatocellular carcinoma (HCC) is currently ranked as the third leading cause of cancer-related mortality worldwide. microRNAs (miRs) serve important roles in the development and progression of HCC. miR-365 has been demonstrated to function as a tumor suppressor in several types of cancer, including HCC; however, the mechanisms by which miR-365 regulates HCC apoptosis remains to be elucidated. In the present study, reverse transcription-quantitative polymerase chain reaction was performed to determine miR-365 expression levels in HCC and normal liver (LO2) cells. miR-365 overexpression was induced in SMC7721 cells using a plasmid-based system, and Cell Counting Kit-8 and TUNEL assays were performed to detect cell activity and apoptosis following miR-365 transfection. A luciferase assay was performed to determine the direct target of miR-365 in apoptosis regulation. Furthermore, a subcutaneously transplanted tumor model was established to evaluate the effects of miR-365 on tumor growth in vivo. The tumor tissue was used for further proliferation and apoptosis detection. The results of the present study indicated that miR-365 expression was significantly lower in HCC cells compared with LO2 cells (P<0.01). Transfection of SMC7721 cells with miR-365 plasmid significantly inhibited cell activity by inducing apoptosis (P<0.01). Luciferase assay indicated that miR-365 targets B-cell lymphoma 2 (Bcl-2) directly and therefore induces the downstream expression of pro-apoptotic proteins. The SMC7721 primary tumor growth was significantly reduced by miR-365 transfection (P<0.01). Further investigation demonstrated that the miR-365 group contained significantly fewer cells that were positive for proliferating cell nuclear antigen (P<0.01) and significantly more apoptotic cells (P<0.01). In conclusion, the results of the present study demonstrated that miR-365 may serve a role in inducing HCC apoptosis via directly targeting Bcl-2. This may provide a novel diagnosis and therapy target for the treatment of patients with HCC.