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Immunotherapy is effective in the treatment of melanoma due to its immunogenicity. In recent years, researchers have identified LTP is rich in active substances that can induce apoptosis in tumor cells and trigger an immune response. However, it is unclear how LTP works to antitumor by enhancing immune cell function. Therefore, the purpose of this study is to investigate the effects of direct and indirect LTP treatments on the quantity and function of Treg cells and CD8+ T cells in B16-F10 melanoma-bearing mice and their associated mechanisms. The results revealed that both direct and indirect LTP treatments were effective at inhibiting tumor growth and extending the survival of B16-F10 melanoma-bearing mice. In addition, both direct and indirect LTP treatments reduced the number of Treg cells and improved the immune function of CD8+ T cells in the spleen and tumor. Nevertheless, the indirect LTP treatment proved to be more effective than the LTP direct treatment. Mechanistic studies demonstrated that direct LTP treatment might affect the quantity and function of Treg cells and CD8+ T cells by modulating cytokine level changes. However, indirect LTP treatment might have a greater tumor immunotherapeutic potential by inhibiting the activation of key protein molecules in the IL-6/STAT3 signaling pathway and regulating cytokine level changes. In conclusion, this study reveals that LTP exerts an anti-melanoma effect by regulating the level and function of Treg cells and CD8+ T cells. This study is expected to provide new ideas for LTP anti-tumor immunotherapy.