Objectives <p>Liver cancer is one of the most fatal malignancies globally, with hepatocellular carcinoma (HCC) accounting for the majority of cases. A hallmark of HCC is its metabolic reprogramming, characterized by aerobic glycolysis (the Warburg effect), which fuels aggressive tumor growth and progression. Recent evidence highlights the role of transcription factors, such as upstream stimulatory factor 1 (USF1), in the metabolic reprogramming of cancer. However, the specific mechanisms by which USF1 regulates metabolism in liver cancer remain to be fully elucidated. Understanding these mechanisms is critical for identifying novel therapeutic targets for the management of&#xa0;HCC.</p> Methods <p>This study utilized hepatocellular carcinoma cell lines (Hep3B and Huh7) and a murine xenograft model to investigate the regulatory role of USF1 in liver cancer metabolism. Functional assays included RNA interference, dual-luciferase reporter assays, ChIP-qPCR, and extracellular flux analysis. Furthermore, data from 374 liver cancer patients were retrospectively&#xa0;analyzed to assess the prognostic significance of USF1.</p> Results <p>Our results demonstrate that USF1 promotes glycolysis in liver cancer by transcriptionally upregulating pyruvate dehydrogenase kinase 1 (PDK1). Knockdown of USF1 reduced glycolysis, lactate production, and ATP levels, while decreasing PDK1 expression. ChIP-qPCR confirmed that USF1 binds to the PDK1 promoter, driving PDK1 expression at the transcriptional level. Moreover, USF1 modulates glycolysis through the Wnt/β-catenin signaling pathway. In vivo experiments showed that silencing USF1 suppresses tumor growth, an effect partially reversed by PDK1 overexpression.</p> Conclusions <p>This study provides the first comprehensive evidence of the USF1–PDK1 axis as a critical regulator of glycolysis and proliferation in hepatocellular carcinoma. Our findings highlight the therapeutic potential of targeting the USF1-PDK1 signaling pathway for the treatment of liver cancer.</p>