Evasion of the immune response is an integral part of the pathogenesis of glioma. In humans, important mechanisms of immune evasion include recruitment of regulatory T cells (Tregs) and polarization of macrophages toward an M2 phenotype. Canine glioma has a robust immune cell infiltrate that has not been extensively characterized. The purpose of this study was to determine the distribution of immune cells infiltrating spontaneous intracranial canine gliomas. Seventy-three formalin-fixed, paraffin-embedded tumor samples were evaluated using immunohistochemistry for CD3, forkhead box 3 (FOXP3), CD20, Iba1, calprotectin (Mac387), CD163, and indoleamine 2,3-dioxygenase (IDO). Immune cell infiltration was present in all tumors. Low-grade and high-grade gliomas significantly differed in the numbers of FoxP3+ cells, Mac387+ cells, and CD163+ cells ( P = .006, .01, and .01, respectively). Considering all tumors, there was a significant increase in tumor area fraction of CD163 compared to Mac387 ( P < .0001), and this ratio was greater in high-grade tumors than in low-grade tumors ( P = .005). These data warrant further exploration into the roles of macrophage repolarization or Treg interference therapy in canine glioma.