Abstract Background Microglia can adopt different morphologies, ranging from a highly ramified to an amoeboid-like phenotype. Although morphological properties of microglia have been described in rodents, little is known about their fine features in humans. The aim of this study was to characterize the morphometric properties of human microglia in gray and white matter of dorsal anterior cingulate cortex (dACC), a region implicated in behavioral adaptation to neuroinflammation. These properties were compared to those of murine microglia in order to gain a better appreciation of the differences displayed by these cells across species. Methods Postmortem dACC samples were analyzed from 11 individuals having died suddenly without any history of neuroinflammatory, neurodegenerative, nor psychiatric illness. Tissues were sectioned and immunostained for the macrophage marker Ionized calcium binding adaptor molecule 1 (IBA1). Randomly selected IBA1-immunoreactive (IBA1-IR) cells displaying features corresponding to commonly accepted microglial phenotypes (ramified, primed, reactive, amoeboid) were reconstructed in 3D and all aspects of their morphologies quantified using the Neurolucida software. The relative abundance of each morphological phenotype was also assessed. Furthermore, adult mouse brains were similarly immunostained, and IBA1-IR cells in cingulate cortex were compared to those scrutinized in human dACC. Results In human cortical gray and white matter, all microglial phenotypes were observed in significant proportions. Compared to ramified, primed microglia presented an average 2.5 fold increase in cell body size, with almost no differences in branching patterns. When compared to the primed microglia, which projected an average of six primary processes, the reactive and amoeboid phenotypes displayed fewer processes and branching points, or no processes at all. In contrast, the majority of microglial cells in adult mouse cortex were highly ramified. This was also the case following a postmortem interval of 43 hours. Interestingly, the morphology of ramified microglia was strikingly similar between species. Conclusions This study provides fundamental information on the morphological features of microglia in the normal adult human cerebral cortex. These morphometric data will be useful for future studies of microglial morphology in various illnesses. Furthermore, this first direct comparison of human and mouse microglia reveals that these brain cells are morphologically similar across species, suggesting highly conserved functions.