OBJECTIVE: Cuproptosis is a novel cell death pathway that was newly discovered in early 2022. However, cuproptosis is still in its infancy in many respects and warrants further research in hepatocellular carcinoma (HCC). This study aimed to analyze the mechanism of cuprptosis in HCC. METHODS: Herein, the tumor microenvironment infiltration landscape of molecular subtypes was illustrated using GSVA, ssGSEA, TIMER, CIBERSORT, and ESTIMATE algorithms based on the expression profile of cuproptosis-related genes (CRGs) from TCGA and GEO databases. Then, the least absolute shrinkage and selection operator regression method was applied to construct a cuproptosis signature to quantify the cuproptosis profile of HCC. Further, we explored the expression of three hub CRGs in cell lines and clinical patient tissues of HCC by Western blotting, qRT-PCR and immunohistochemistry. Finally, we examined the function of dihydrolipoamide S-acetyltransferase (DLAT) in cuproptosis in HCC by loss-of-function strategy, Western blotting and CCK8 assay. RESULTS: Three distinct molecular subtypes were identified. Cluster 2 had the greatest infiltration of immune cells with best prognosis. The cuproptosis signature was indicative of tumor subtype, immunity, and prognosis for HCC, and specifically, a low cuproptosis score foreshadowed good prognosis. DLAT was highly expressed in liver cancer cell lines and HCC tissues and positively correlated with clinical stage and grade. We also found that potent copper ionophore elesclomol could induce cuproptosis in a copper-dependent manner. Selective Cu chelator ammonium tetrathiomolybdate and downregulating DLAT expression by siRNA could effectively inhibit cuproptosis. CONCLUSION: Cuproptosis and DLAT as a promising biomarker could help to determine the prognosis of HCC and may offer novel insights for effective treatment.