Guanosine, a guanine-based purine, is an extracellular signaling molecule that is released from astrocytes and shows neuroprotective effects in several in vivo and in vitro studies. Our group recently showed that guanosine presents antioxidant properties in C6 astroglial cells. The heme oxygenase 1 (HO1) signaling pathway is associated with protection against oxidative stress. Azide, an inhibitor of the respiratory chain, is frequently used in experimental models to induce oxidative and nitrosative stress. Thus, the goal of this study was to investigate the effect of guanosine on azide-induced oxidative damage in C6 astroglial cells. Azide treatment of these cells resulted in several detrimental effects, including induction of cytotoxicity and mitochondrial dysfunction, increased levels of reactive oxygen/nitrogen species, iNOS expression and NADPH oxidase, decreased glutamate uptake and EAAC1 glutamate transporter expression, decreased glutathione (GSH) levels, and decreased activities of glutamine synthetase (GS), superoxide dismutase (SOD) and catalase (CAT). The treatment also increased NFκB activation and the release of proinflammatory cytokines TNF-α and IL-1β. Guanosine strongly prevented these effects, protecting glial cells against azide-induced cytotoxicity and modulating glial, oxidative and inflammatory responses through the activation of the HO1 pathway. These observations reinforce and support the role of guanosine as an antioxidant molecule against oxidative damage. This article is protected by copyright. All rights reserved.