BACKGROUND: Vitex negundo L (Verbenaceae) is an aromatic shrub that is abundant in Asian countries. A series of compounds from Vitex negundo have been used in traditional Chinese medicine for the treatment of various diseases. Cutaneous melanoma is one of the most aggressive malignancies. A significant feature of melanoma is its resistance to traditional chemotherapy and radiotherapy; therefore, there is an urgent need to develop novel treatments for melanoma. METHODS: We first examined the effects of VB1 (vitexin compound 1) on cell viability by CCK-8 (cell counting kit) and Colony Formation Assay; And then, we analyzed the apoptosis and cell cycle by flow cytometry, verified apoptosis by Immunoblotting. The in vivo effect of VB1 was evaluated in xenograft mouse model. Potential mechanisms of VB1's antitumor effects were explored by RNA sequencing and the key differential expression genes were validated by real-time quantitative PCR. Finally, the intracellular reactive oxygen species (ROS) level was detected by flow cytometry, and the DNA damage was revealed by Immunofluorescence and Immunoblotting. RESULTS: In this study, we show that VB1, which is a compound purified from the seed of the Chinese herb Vitex negundo, blocks melanoma cells growth in vitro and in vivo, arrests the cell cycle in G2/M phase and induces apoptosis in melanoma cell lines, whereas the effects are not significantly observed in normal cells. To study the details of VB1, we analyzed the alteration of gene expression profiles after treatment with VB1 in melanoma cells. The findings showed that VB1 can affect various pathways, including p53, apoptosis and the cell cycle pathway, in a variety of melanoma cell lines. Furthermore, we confirmed that VB1 restored the P53 pathway protein level, and then we demonstrated that VB1 significantly induced the accumulation of ROS, which resulted in DNA damage in melanoma cell lines. Interestingly, our results showed that VB1 also increased the ROS levels in BRAFi (BRAF inhibitor)-resistant melanoma cells, leading to DNA cytotoxicity, which caused G2/M phase arrest and apoptosis. CONCLUSIONS: Taken together, our findings indicate that vitexin compound 1 might be a promising therapeutic Chinese medicine for melanoma treatment regardless of BRAFi resistance.