Hepatocellular carcinoma (HCC) is a highly aggressive and highly malignant cancer. Glucose metabolic reprogramming provides sufficient ATP to support HCC's rapid proliferation and invasion. Consequently, this study intends to investigate the effects of FAM99A and FAM99B on glucose metabolic reprogramming, and provide new insights for HCC treatment. Changes in malignant phenotypes and glycolysis-related indices of HCC cells (HCCLM3 and HEPG2) were assessed after exogenous regulation of FAM99A and FAM99B under hypoxic conditions. Oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and glycolytic proton efflux rate (glycoPER) were measured using the Seahorse XF Glycolysis Rate Assay Kit (103344-100, Agilent). HCCLM3 cells were subjected to transcriptome and smallRNA sequencing to identify differentially expressed genes (DEGs) and miRNAs (DE-miRNAs) associated with FAM99A and FAM99B. Under hypoxic conditions, the expression of FAM99A and FAM99B was significantly downregulated in HCC cells. Overexpression of FAM99A or FAM99B significantly inhibited HCC cell proliferation, wound healing, and invasion. Moreover, they effectively decreased intracellular glucose, extracellular lactate, ATP, glycolysis-related enzymes, ECAR, and glycoPER, and increased pH, extracellular glucose, and mitoOCR/glycoPER. A total of 31 DEGs and 15 DE-miRNAs were present in HCCLM3 cells overexpressing FAM99A, and 375 DEGs and 68 DE-miRNAs were identified in HCCLM3 cells overexpressing FAM99B. These DEGs and DE-miRNA targets were involved in cell cycle, apoptosis, metastasis, extracellular matrix remodeling, and metabolic reprogramming. The FAM99B-associated ceRNA network contained one DE-miRNA and 10 DEGs, and their expression differences were consistent with the sequencing results. Hypoxia-induced suppression of FAM99A and FAM99B facilitates proliferation, metastasis, and glucose metabolic reprogramming of HCC.© 2025 The Author(s). The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.