Despite rapid developments in immunotherapy and targeted therapy, dacarbazine (DTIC)-based chemotherapy still has been placed at the first-line for advanced melanoma patients who are after failure of immunotherapy or targeted therapy. However, the limited response rate and survival benefit challenge the DTIC-based chemotherapy for advanced melanoma patients.Two melanoma cell lines, A375 and SK-MEL-28 were cultured with PA and DTIC over a range of concentrations for 72 h and the cell viabilities were detected by CCK8 assay. The Bliss model and ZIP model were used for calculating the synergistic effect of PA and DTIC. DNA double-strand breaks in the two cell lines were examined by the Comet assay, and cell apoptosis was analyzed by flow cytometry. The short hairpin RNA (shRNA)-mediated knockdown, Real-time polymerase chain reaction (RT-PCR) and Western blot were performed for molecular analysis.In the present study, we report that Protocatechuic aldehyde (PA) synergistically enhances the cytotoxicity of DTIC to two melanoma cell lines, A375 and SK-MEL-28. The combination of PA and DTIC augments DNA double-strand breaks and increases cell apoptosis. Further mechanism study reveals that PA destabilizes MGMT protein (O-6-Methylguanine-DNA Methyltransferase) through the ubiquitin-proteasome process and directly repairs DTIC-induced genetic lesions. Knockdown of MGMT compromises the synergistic effect between PA and DTIC.Our study demonstrates that the bioactive compound, Protocatechuic aldehyde, synergistically promotes the cytotoxicity of DTIC to melanoma cells through destabilization of MGMT protein. It could be a potential candidate for melanoma chemotherapy.© 2023. The Author(s).