Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related mortality worldwide. Despite the availability of first-line therapies, the median survival time for patients with advanced HCC remains only 6–10 months. Poor drug responsiveness and high recurrence rates highlight the urgent need to identify new therapeutic targets for HCC. The tumor invasive margin, situated at the interface between tumor and surrounding liver tissue, captures early tumorigenic events and metabolic rewiring associated with cancer progression. Using label-free LC–MS proteomics on patient-derived liver tissues from distal, margin, and tumor regions, we identified GTP cyclohydrolase I feedback regulatory protein (GCHFR) as a margin-enriched protein signature. GCHFR regulates GTP cyclohydrolase I (GCH1) activity in a tetrahydrobiopterin (BH₄)-dependent manner: when BH₄ levels are high, GCHFR forms an inhibitory complex with GCH1, whereas under low BH₄ conditions, GCHFR stabilizes the active GCH1 conformation to restore BH₄ and promote nitric oxide synthesis. However, the role of GCHFR in HCC progression and metabolism remains unexplored. Using shRNA-mediated knockdown in HepG2 cells, we show that silencing GCHFR significantly reduces cell proliferation, migration, and invasion, without major alterations in fatty acid or glucose metabolism. Moreover, GCHFR silencing increases the sensitivity of HCC cells to the chemotherapeutic agent etoposide. Integrated proteomic analysis further revealed a marked downregulation of pathways associated with cell cycle progression. Together, this study identifies GCHFR as a novel regulator of cell cycle control and a potential therapeutic target for HCC.