Astrocytes and microglial cells play an important role in the homeostasis, detoxification and immune response against chemical, infectious or traumatic challenges in the Central Nervous System (CNS). These cells react to external insults by changes in their morphology, and by neurotrophic and/or neurotoxic factors release. Rutin is a natural flavonoid used in popular medicine in Brazil, but its effects on CNS cells is poorly known. This study investigated the effects of rutin, in vitro, on astrocyte/microglial primary mixed cultures, derived from neonatal rat cortical brains. The cultures were treated for 24h, with rutin (50µM and 100µM) or vehicle (DMSO 0,5%). No changes on mitochondrial activity, revealed by the MTT test, were detected for any treatment. However, an increase on LDH activity was evident on culture medium of the glial cells treated with 100µM rutin, suggesting a membrane instability. Astrocytes exposed to the flavonoid rutin presented structural changes: with 50mM rutin astrocytes began to retract the cell body and emitted short and thick processes; with 100mM rutin these cells showed stellar phenotype which presented a reduction in GFAP levels. Such modifications were revealed by Rosenfeld’s staining, immunocytochemistry and western immunoblotting for GFAP and OX-42 (astrocyte and microglia markers, respectively). Microglia appears as macrophagic cells, and an increase in the proportion of OX-42 positive microglial cells was observed, with values reaching 12% and 18% in cultures treated with 50 and 100mM rutin, respectively, compared with 4% found in controls. Moreover, a significant (P< 0.05) increase on the levels of nitrite, which expressed the NO production, was observed on cultures exposed to 100µM rutin. Furthermore, we also observed a significant increase (P< 0.05) on the levels of TNFa in the culture medium of primary cultures exposed to both 50 and 100mM rutin. Taken together, these results demonstrated that the flavonoid rutin promotes glial cells activation with changes in cell morphology and interferes on NO and TNFa release.