Melatonin, also known as the pineal hormone, is secreted by the pineal gland and primarily regulates circadian rhythms. Additionally, it possesses immunomodulatory properties and anticancer effects. However, its specific mechanism in hepatocellular carcinoma (HCC) remains unclear, particularly regarding its effect on HCC-mediated immune escape through PD-L1 expression.In this study, in vitro experiments were conducted using Huh7 and HepG2 HCC cells. Melatonin treatment was applied to both cell types to observe changes in malignant phenotypes. Additionally, melatonin-pretreated Huh7 or HepG2 cells were co-cultured with T cells to simulate the tumor microenvironment. The results showed that melatonin inhibited cancer cell proliferation, migration, and invasion, as well as reduced PD-L1 expression in cancer cells, exhibiting similar anti-cancer effects in the co-culture system. In vivo experiments involved establishing ascitic HCC mouse models using H22 cells, followed by subcutaneous tumor models in Balb/c nude and Balb/c wild-type mice. Melatonin inhibited tumor growth and suppressed PD-L1 expression in cancer tissues in both subcutaneous tumor models, and it increased T lymphocyte activity in the spleen of Balb/c wild-type mice. Overall, the in vitro and in vivo experiments demonstrated that melatonin has dual anti-cancer effects in HCC: direct intrinsic anti-cancer activity and enhancement of anti-tumor immunity by reducing PD-L1 expression thereby inhibiting cancer immune escape. Furthermore, a decrease in the expression of the upstream molecule HIF-1α of PD-L1 and an increase in the expression levels of JNK, P38, and their phosphorylated forms were detected. Thus, the mechanism by which melatonin reduces PD-L1 may involve the downregulation of HIF-1α expression or the activation of the MAPK-JNK and MAPK-P38 pathways. This provides new insights and strategies for HCC treatment.