BACKGROUND: Reports show miR-23b to be a cancer-related biomarker in various cancer types. Interestingly, it has a dual role of oncogenic and tumor-suppressive functions, depending on the cancer type. This study focused on the unknown association of miR-23b-3p with hepatocellular carcinoma (HCC). METHODS: Expression of miR-23b-3p was measured in nine HCC cell lines and 125 resected human HCC samples by TaqMan microRNA assays. To detect its downstream target, miR-23b-3p mimic and inhibitor constructs were transfected and analyzed. RESULTS: HepG2, a high miR-23b-3p-expressing cell line, was transfected with a miR-23b-3p inhibitor construct, whereas SK-Hep1, a low miR-23b-3p-expressing cell line, was transfected with a mimic construct. Proliferation of HCC cells was activated by miR-23b-3p overexpression and diminished by its knockdown. Then, 125 clinical HCC samples were examined to measure miR-23b-3p expression. Tumor expression of miR-23b-3p was upregulated in 48 cases (38%) and downregulated in 77 cases (62%). The upregulated cases were correlated with elderly patients (P = 0.015). These patients also showed significantly poor overall survival [hazard ratio (HR), 3.10; 95% conflidence interval (CI), 1.57-6.29; P = 0.001] in a multivariate analysis. Furthermore, mitochondrial metabolism-related genes (MICU3 and AUH) were detected as specific binding targets. CONCLUSION: The study showed that miR-23b-3p functions as an oncogenic microRNA in HCC cell lines. Its overexpression in resected HCC tissues was a significant prognostic factor of overall survival. Both MICU3 and AUH may be candidate gene targets of miR-23b-3p.