The regional heterogeneity of neuronal phenotypes is a well-known phenomenon. Whether or not glia derived from different brain regions are phenotypically and functionally distinct is less clear. Here, we show that microglia, the resident immune cells of the brain, display region-specific responses for activating agents including glutamate (GLU), lipopolysaccharide (LPS) and adenosine 5′-triphosphate (ATP). Primary microglial cultures were prepared from brainstem (Brs), cortex (Ctx), hippocampus (Hip), striatum (Str) and thalamus (Thl) of 1-day-old rats and were shown to upregulate the release of nitric oxide (NO) and brain-derived neurotrophic factor (BDNF) in a region- and activator-specific manner. With respect to ATP specifically, ATP-induced changes in microglial tumor necrosis factor-α (TNF-α) release, GLU uptake and purinergic receptor expression were also regionally different. When co-cultured with hypoxia (Hyp)-injured neurons, ATP-stimulated microglia from different regions induced different levels of neurotoxicity. These region-specific responses could be altered by pre-conditioning the microglia in a different neurochemical milieu, with taurine (TAU) being one of the key molecules involved. Together, our results demonstrate that microglia display a regional heterogeneity when activated, and this heterogeneity likely arises from differences in the environment surrounding the microglia. These findings present an additional mechanism that may help to explain the regional selectiveness of various brain pathologies.