BACKGROUND: Cuproptosis is a new type of programmed cell death involved in the regulation of neuroendocrine tumors, immune microenvironment, and substance metabolism. However, the role of cuproptosis-related genes (CRGs) in Hepatocellular carcinoma (HCC) remains unclear. METHOD: Through multiple bioinformatics analysis, we constructed a prognostic gene model and competing endogenous RNA (ceRNA) network. The correlation between CRGs and prognosis, immune infiltration, immune checkpoints, microsatellite instability (MSI) and tumor mutational burden (TMB) was analyzed by Kaplan-Meier curve, univariate Cox, multivariate regression, and Spearman's analysis in HCC patients. Besides, the qRT-PCR and immunohistochemistry assays were used to determine prognostic CRGs mRNA and protein expression in HCC. RESULTS: We established a novel 3-gene signature related to CRGs for evaluating the prognosis of HCC patients. HCC patients with high risk scores had a poor prognosis with an area under the curve of 0.737, 0.646, and 0.634 on 1-year, 3-year, and 5-year receiver operating characteristic curves. Significant correlation was observed between prognostic CRGs and immune infiltration, immune checkpoints, MSI and TMB. We also developed five ceRNA networks to regulate the occurrence and progression of HCC. CDKN2A, DLAT, and PDHA1 protein expression was up-regulated in HCC versus normal tissues. Besides, the mRNA expression levels of CDKN2A, DLAT, GLS, and PDHA1 were elevated in the HCC cell lines compared to the normal liver cell lines. CONCLUSIONS: This novel prognostic CRGs signature could be accurately predict the prognosis of patients with HCC. The ceRNA regulatory network might be potential prognostic biomarkers and therapeutic targets for HCC patients.