AbstractHepatocellular carcinoma (HCC) is characterised by a hypoxic microenvironment and a high rate of heterogeneity and recurrence, and the presence of cancer stem cells (CSCs) in HCC may well explain both of these pathological properties. There is mounting evidence that long non-coding RNAs (lncRNAs) participate in carcinogenesis and maintain cancer stemness of HCC cells. However, the expression modes, regulatory mechanisms and potential roles of stemness-related lncRNAs in HCC are still obscure. LncRNA RUNX1-IT1 is the intronic transcript 1 of the RUNX1, which is also known as chromosome 21 open-reading frame 96 (C21orF96). Although the functions of the RUNX1 have been identified in different diseases, the function and its potential mechanisms of the lncRNA RUNX1-IT1 in HCC still remains to be largely unknown. In this study, we verified that the expression of LncRNA RUNX1-IT1 was decreased in GEO data set, HCC samples and correlated with unfavourable clinicopathologic characteristics and poor prognosis. RUNX1-IT1 repressed HCC cell proliferation, cell cycle progression, invasion and cancer stemness and induced apoptosis in vitro. Overexpression of RUNX1-IT1 impaired the growth, metastasis and stem-like features of HCC cells in vivo. Mechanistically, RUNX1-IT1 directly bound to miR-632 and acted as competing endogenous RNA to facilitate the expression of the miR-632 target gene GSK-3β and subsequently modulate the WNT/β-catenin pathway in HCC cells. Furthermore, hypoxia-driven histone deacetylase 3 (HDAC3), as an upstream regulatory mechanism, was critical for the downregulation of RUNX1-IT1 in HCC. Thus, lncRNA RUNX1-IT1, as a regulator of hypoxia, may function as a potential therapeutic target for conquering HCC.