Induction of cuproptosis in tumor cells is an emerging direction for cancer drug development. Plumbagin (PLB), a natural biological molecule, has anticancer activities, partially via copper-dependent mechanisms. But it remains unclear if PLB can induce cuproptosis in hepatocellular carcinoma (HCC). In this study, PLB showed HCC-suppressive activities and caused representative molecular phenotypes of cuproptosis, whereas tetrathiomolybdate, an inhibitor of cuproptosis, could alleviate these effects the most. The mRNA and protein expression levels of the primary hepatic copper exporter, ATPase copper transporting beta (ATP7B), decreased in PLB-treated HCC cells, which might cause the accumulation of intracellular copper and trigger cuproptosis. An upstream ATP7B-regulatory microRNA, microRNA-302a-3p (miR-302a-3p), was identified by quantification and validated by the overexpression/inhibition experiment and luciferase reporter assay. Moreover, PLB was found to reduce the protein level of DNA-methyltransferase 1 (DNMT1), thereby enhancing the promoter hypomethylation and the expression of miR-302a-3p. Gene manipulation experiments further demonstrated that ATP7B, miR-302a-3p, and DNMT1 mediated PLB-induced cuproptosis. Preliminary clinical analyses showed that low ATP7B expression levels were associated with better prognosis, supporting the importance of ATP7B-lowering therapeutic strategies in HCC. Together, our results indicate that PLB triggers HCC cuproptosis via the DNMT1/miR-302a-3p/ATP7B axis, providing a potential therapeutic strategy for HCC.