Melanoma is among the most common malignancies and has recently exhibited increased resistance to treatments, resulting in a more aggressive disease course. Mesenchymal stem cells (MSCs) secrete cytokines both in vivo and in vitro, which regulate tumor cell signaling pathways and the tumor microenvironment, thereby influencing tumor progression. This study investigates the anti-melanogenesis effects of sheep umbilical cord mesenchymal stem cells (SUCMSCs) to assess their potential application in melanoma treatment. Our findings indicate that, in vitro, SUCMSCs reduce melanin content and tyrosinase activity, inhibit melanoma cell viability, proliferation, migration, and invasion, and promote melanoma cell apoptosis. Subsequent in vivo experiments confirmed that SUCMSCs effectively suppress tumor growth, and histological analysis via HE staining revealed notable differences. Additionally, transcriptome sequencing analysis indicated that the anti-tumor effects were primarily mediated through autophagy, apoptosis, and the TGF-β and NF-κB signaling pathways. The RT-qPCR validation results aligned with the transcriptome data. In summary, SUCMSCs exert anti-melanogenesis effects through the interaction of multiple signaling pathways and cytokines, demonstrating significant potential for melanoma treatment.