Background: Melanoma is one of the most aggressive malignancies. Drug resistance and toxicity limits the clinical efficacy of melanoma chemotherapeutic drugs such as dacarbazine. Therefore, the development of chemotherapeutic agents for melanoma treatment is urgently needed. Methods: The cytotoxicity of RJT-101 on melanoma was evaluated by MTS assay. Cell cycle arrest, apoptosis, and DNA damage in melanoma cells in vitro were measured by flow cytometry and Western blotting. In addition, the apoptosis and DNA damage of melanoma cells was measured by TUNEL assay and Immunofluorescence, respectively. Gene expression was assessed by rt-PCR. The antitumor efficacy of RJT-101 in vivo was evaluated by mouse models. The underlying molecular mechanisms of Top1 function were measured by DNA unwinding assay, immunoprecipitation and Western blotting. Findings: RJT-101 significantly inhibits the proliferation of melanoma cells, however has low cytoxicity to non-malignant cells. RJT-101 induces apoptosis, DNA damage, and G2/M phase arrest, as a consequent, attenuates tumor growth, lung metastasis in vivo as well as prolongs survival of tumor bearing mice. RJT-101 could block Top1 activity as well as induce its degradation through proteasome system. Interestingly, Top1 is over-expressed in melanoma cells, compared to non-malignant cells. Knock down of Top1 suppresses melanoma cells growth and induces apoptosis and DNA damage in melanoma cells. Interpretation: RJT-101 effectively inhibits melanoma cells (including vemurafenib-resistant melanoma cells) proliferation in vitro and in vivo through the induction of DNA damage and apoptosis by inhibiting of Top1, indicating RJT-101 is a promising chemotherapeutic agent which warrants further clinical evaluation. Funding Statement: This work was supported by Major Projects of International Cooperation and Exchanges NSFC Grand No.81620108024 and Grant No. 81572679, 81472852. and the Distinguished Professor Research Startup Funding (S. Cao) from Southwest Medical University (2015-RCYJ0002). The Strategy-Oriented Special Project of Central South University in China(ZLXD2017003). Declaration of Interests: The authors declared: RJT-101 was synthesized by Chifeng Saliont Pharmaceuticals Co. Ltd. The anticancer effects of RJT-101 and the potential mechanisms of RJT-101 associated with its anti-melanoma activity were evaluated and studied by the Department of Dermatology of Xiangya Hospital, Hunan Key Laboratory of Skin Cancer and Psoriasis and Hunan Engineering Research Center of Skin Health and Disease. There are no competing interests between the synthesis of RJT-101 and the study of RJT-101 in the inhibition of melanoma. Ethics Approval Statement: All animal experiments were carried out following a protocol approved by the Committee on Use and Care of Animal of Xiangya Hospital, Central South University (Changsha, Hunan, China).