Objective To investigate the effect of long non-coding RNA UPK1A-AS1 on glycolysis of hepatocellular carcinoma cells and its underlying molecular mechanisms. Methods A hepatocellular carcinoma (HCC) cell line with lentivirus-mediated stable UPK1A-AS1 overexpression and the cells infected with a negative control lentiviral vector were cultured under normoxic (21% O2) or hypoxic (1% O2) conditions for 24 h. The effect of UPK1A-AS1 overexpression on glycolysis of the HCC cells was examined. The expressions of glycolysis-related genes HIF1A, GLUT1, HK1, HK2 and PGK1 were detected by qRT-PCR, and the effect of UPK1A-AS1 overexpression on HRE activity was detected by dual luciferase report assay. The HCC cells were treated with cycloheximide to detect the effect of UPK1A-AS1 overexpression on the stability of HIF-1α protein. Immunoprecipitation assay was used to analyze the changes in ubiquitin modification of HIF-1α protein in response to UPK1A-AS1 overexpression. The effects of UPK1A-AS1 overexpression and RNA interference of HIF-1α expression on glucose consumption, lactate production and expressions of HRE activity and glycolysis- related genes (HK1, HK2 and PGK1) were examined in the HCC cells. Results Compared with the control group, overexpression of UPK1A-AS1 significantly promoted glucose consumption and lactate production in HCC cells under both normoxic and hypoxic conditions (P<0.05). Overexpression of UPK1A-AS1 significantly increased the expression of glycolysis-related genes including HIF1A, GLUT1, HK1, HK2 and PGK1. Upregulation of UPK1A-AS1 obviously promoted the transcriptional activity of HRE (P<0.05). Western blotting showed that UPK1A-AS1 overexpression obviously increased the stability of HIF-1α protein and significantly reduced ubiquitin modification of HIF-1α. Downregulation of HIF-1α obviously reversed the effect of UPK1A-AS1 overexpression in promoting glucose consumption, lactate production and HRE luciferase activity. Silencing HIF-1α also suppressed the upregulation of glycolysis-related gene expressions induced by UPK1A-AS1 overexpression (P<0.05). Conclusion The long non-coding RNA UPK1A-AS1 upregulates the expression of glycolysis-related genes by stabilizing the expression of HIF-1α, thereby promoting glycolysis level in HCC cells.