Melanoma in humans and canines is an aggressive and highly metastatic cancer. The mucosal forms in both species share genetic and histopathologic features, making dogs a valuable spontaneous disease animal model. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells of myeloid origin with immunosuppressive capabilities, which are increased in many human cancers and contribute to tumor immune evasion. They are a possible target to improve immunotherapy outcomes. Current information regarding MDSCs in canines is minimal, limiting their use as translational model for the study of MDSCs. The objective of this study was to characterize major MDSCs subsets (monocytic and polymorphonuclear) and the cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 10 (IL-10) and monocyte chemoattractant protein-1 (MCP-1) in canines with malignant melanoma and to evaluate changes in MDSCs and the cytokines over time in response to a GD3-based active immunotherapy. Whole blood and serum collected from 30 healthy controls and 33 patients enrolled in the University of Florida melanoma vaccine trial were analyzed by flow cytometry with canine specific CD11b, MHCII and anti-human CD14 antibodies to assess ostensibly polymorphonuclear-MDSC (CD11b+ MHCII- CD14-) and monocytic-MDSC (CD11b+ MHCII- CD14+) subsets. IL-10, MCP-1 and both MDSCs subsets were significantly elevated in melanoma dogs versus controls. Both MDSCs subsets decreased significantly following GD3-based immunotherapy administration but no significant changes in cytokines were seen over time. To our knowledge, this is the first report documenting increased monocytic-MDSCs in canine melanoma. This is consistent with human malignant melanoma data, supporting dogs as a valuable model for therapeutic intervention studies. Copyright © 2019 Elsevier B.V. All rights reserved.