<strong>BACKGROUND</strong> Melanoma is among the most aggressive forms of cancer. Our latest retrospective analysis showed that recombinant human interferon-alpha1b (IFN-alpha1b) led to significantly prolonged survival with mild toxicity in patients with stage IV melanoma. Based on this clinical finding, the current study sought to investigate the influence of IFN-alpha1b on the antitumor immunity of melanoma, with interferon-alpha2b (IFN-alpha2b) used as a control. <strong>MATERIAL AND METHODS</strong> Peripheral blood mononuclear cells were stimulated with culture medium alone, or medium supplemented with IFN-alpha1b or IFN-alpha2b. Flow cytometry and lactate dehydrogenase release assays were used to evaluate cytotoxic effects. Flow cytometry and enzyme-linked immunospot assays were used to analyze immunoregulatory effects on natural killer (NK) cells, natural killer T (NKT) cells, CD3⁺CD8⁺ T cells, and melanoma cells. Cell Counting Kit-8 assay was performed to measure the effect on proliferation of melanoma cells in vitro. <strong>RESULTS</strong> IFN-alpha1b enhanced the activity of NK cells, NKT cells, and CD3⁺CD8⁺ T cells from melanoma patients. Compared with IFN-alpha2b, IFN-alpha1b induced a relatively lower level of programmed cell death-ligand 1 (PD-L1) in melanoma cells without affecting the expression of PD-L1 in CD3⁺CD8⁺ T cells. Additionally, IFN-alpha1b showed a much stronger inhibition of the proliferation of melanoma cells than IFN-alpha2b. <strong>CONCLUSIONS</strong> IFN-alpha1b has an immunostimulatory activity similar to IFN-alpha2b and possesses milder adverse effects on immune checkpoints and stronger inhibitory effects on melanoma cell growth than IFN-alpha2b. Therefore, IFN-alpha1b is a promising drug for the treatment of melanoma.