Hepatocellular carcinoma (HCC) is the most common type of liver cancer. The stemness of cancer cells and hypoxic microenvironment in HCC influence tumor progression and resistance to anti-tumor therapies. Herein, we aimed to combine tumoral stemness and hypoxia features to evaluate the prognosis and tumor immune microenvironment (TIME) in HCC.Based on the HCC data from The Cancer Genome Atlas (TCGA) database, the mRNA expression-based stemness index (mRNAsi) was calculated, followed by acquisition of stemness-hypoxia genes. The prognostic stemness-hypoxia genes were identified using Cox regression analysis and a stemness-hypoxia prognostic model was constructed. The prognostic capacity of the model was validated, and the associations between the model and immune characteristics were evaluated. Moreover, the differential expression of model genes in HCC cells under hypoxia condition was determined.A three-gene prognostic signature based on the stemness-hypoxia genes (PPARGC1A, PFKFB3, and SLC2A5) was constructed. The Kaplan-Meier curve validated the prognostic capacity of the model. PPARGC1A and PFKFB3 were independent prognostic factors for HCC: PPARGC1A expression was significantly associated with favorable overall survival (OS) of HCC patients, while PFKFB3 expression was related to poor OS. Furthermore, the high-risk group was associated with advanced stages, infiltrated tumor-promoting immune cells, and elevated expression of immune checkpoints. The risk score also exhibited prognostic capacity for the OS and predictive value for the immune microenvironment in HCC. Finally, SLC2A5 was significantly up-regulated in HepG2 cells compared to LO-2 cells. Additionally, elevated SLC2A5 expression and reduced PPARGC1A and PFKFB3 expression were observed in both Hep3B and HepG2 cells under hypoxia condition.Our established stemness-hypoxia gene signature showed favorable prognosis performance for HCC and was related to TIME. Our findings provide novel insights into the prognostic evaluation of HCC.Copyright © 2025 AME Publishing Company. All rights reserved.