Copper (Cu) is an essential trace element and acts as a redox cofactor for many enzymes; however, excess Cu is toxic to cells. Hydrogen sulfide (H₂S) is a well-known toxic gaseous molecule, but it has various biological effects such as neuromodulation and vasodilation. H₂S was recently demonstrated to be involved in the detoxification of heavy metals, including zinc and cadmium, suggesting that H₂S helps to maintain the homeostasis of heavy metals in cells. However, it is unclear how H₂S impacts cellular Cu dynamics. In this study, we examined the effects of H₂S on Cu cytotoxicity. Human neuroblastoma SH-SY5Y cells were exposed to CuSO₄ in the presence of the H₂S donor NaHS. CuSO₄ alone slightly induced cell injury, whereas the combination of CuSO₄ and NaHS (Cu/NaHS) increased Cu cytotoxicity. The Cu chelator bathocuproinedisulfonic acid mitigated Cu/NaHS-induced cytotoxicity. Compared with CuSO₄ alone, Cu/NaHS markedly promoted ROS generation, mitochondrial dysfunction, and a decrease in ATP production. In addition, reporter assay using the metal responsive element (MRE)-driven reporter plasmid revealed that Cu/NaHS augmented Cu-dependent MRE activation. The amount of intracellular Cu was significantly higher in cells treated with Cu/NaHS than in those treated with CuSO₄ alone. Moreover, Cu/NaHS markedly suppressed the level of the Cu exporter ATP7A, but not ATP7B, protein, whereas the combination did not affect that of the Cu importer CTR1 protein. Taken together, we conclude that the marked decrease in the ATP7A protein level by Cu/NaHS promotes intracellular Cu accumulation and leads to increased Cu cytotoxicity.