Insights into the role of the mitogen-activated protein kinase (MAPK) pathway and immune checkpoints have led combined targeted therapy and immunotherapy to be a promising regimen. Trametinib, as a mitogen-activated extracellular signal-regulated kinase (MEK) inhibitor, has demonstrated effectiveness in patients with advanced melanoma. T cell immunoglobulin-and mucin-domain-containing molecule-3 (Tim-3), an immune checkpoint molecule, participates in multiple negative regulation of antitumor immunity. We for the first time to our knowledge reported the combination of trametinib and anti-Tim-3 monoclonal antibody (mAb) in treating B16-F10 melanoma mice. We discovered that trametinib remarkably promoted apoptosis and inhibited cell proliferation while inhibition of MEK improved the expression of Tim-3 and caused the decrease of CD8(+) T cells; to the contrary, anti-Tim-3 mAb enhanced antitumor immunity by stimulating CD8(+) T cells, thus the combined therapy produced potent antitumor effect cooperatively. Taken together, our study provides compelling evidence for combining trametinib and anti-Tim-3 mAb as a potential valuable regimen in treating melanoma. (C) 2017 Elsevier Inc. All rights reserved.