The regional heterogeneity of microglia was first described a century ago by Pio del Rio Hortega. Currently, new information on microglia heterogeneity throughout central nervous system (CNS) regions is being revealed by high-throughput techniques. It remains unclear whether these spatial specificities translate into different microglial behaviors in vitro. We cultured microglia isolated from the cortex and spinal cord and analyzed the effect of the CNS spatial source on behavior in vitro by applying the same experimental protocol and culture conditions. We analyzed the microglial cell numbers, function, and morphology and found a distinctive in vitro phenotype. We found that microglia were present in higher numbers in the spinal-cord-derived glial cultures, presenting different expressions of inflammatory genes and a lower phagocytosis rate under basal conditions or after activation with LPS and IFN-γ. Morphologically, the cortical microglial cells were more complex and presented longer ramifications, which were also observed in vivo in CX3CR1+/GFPtransgenic reporter mice. Collectively, our data demonstrated that microglial behavior in vitro is defined according to specific spatial characteristics acquired by the tissue. Thus, our study highlights the importance of microglia as a source of CNS for in vitro studies.