Background: MicroRNAs (miRNAs) play crucial roles in the development of hepatocellular carcinoma (HCC). Hsa-microRNA-27b-3p (hsa-miR-27b) is involved in the formation and progression of various cancers, but its role and clinical value in HCC remain unclear. Methods: The expression of hsa-miR-27b in HCC was examined by quantitative real-time PCR (qRT-PCR) and in situ hybridization (ISH) assays of clinical samples. Cell Counting Kit-8 assays (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU) incorporation assays, Transwell assays, filamentous actin (F-actin) staining and western blot analyses were used to determine the effects of hsa-miR-27b on HCC cells in vitro. Subcutaneous xenograft and lung metastatic animal experiments were conducted to verify the role of hsa-miR-27b in HCC in vivo. In silico prediction, qRT-PCR, western blot, anti-Argonaute 2 (AGO2) RNA immunoprecipitation (RIP) and dual luciferase reporter assays were applied to identify the target genes of hsa-miR-27b. To detect the impacts of hsa-miR-27b on nuclear factor kappa B (NF-kappa B) signalling cascades mediated by transforming growth factor-activated kinase-binding protein 3 (TAB3), we performed qRT-PCR, western blot assays, immunofluorescence staining, immunohistochemistry (INC) and dual-luciferase reporter assays. Recombinant oncolytic adenovirus (Onco(Ad)) overexpressing hsa-miR-27b was constructed to detect their therapeutic value in HCC. Results: The expression of hsa-miR-27b was lower in HCC than in adjacent nontumourous tissues (ANTs), and the reduced expression of hsa-miR-27b was associated with worse outcomes in patients with HCC. Hsa-miR-27b significantly inhibited the proliferation, migration, invasion, subcutaneous tumour growth and lung metastasis of HCC cells. The suppression of hsa-miR-27b promoted the nuclear translocation of NE-kappa B by upregulating TAB3 expression. TAB3 was highly expressed in HCC compared with ANTs and was negatively correlated with the expression of hsa-miR-27b. The impaired cell proliferation, migration and invasion by hsa-miR-27b overexpression were recovered by ectopic expression of TAB3. Recombinant Onco(Ad) with overexpression of hsa-miR-27b induced anti-tumour activity compared with that induced by negative control (NC) Onco(Ad) in vivo and in vitro. Conclusions: By targeting TAB3, hsa-miR-27b acted as a tumour suppressor by inactivating the NF-kappa B pathway in HCC in vitro and in vivo, indicating its therapeutic value against HCC.