Guanosine (GUO) is an endogenous modulator of glutamatergic excitotoxicity and promotes neuroprotection in in vitro and in vivo models of neurotoxicity. Guanosine promotes neuroprotection against oxygen and glucose deprivation (OGD) by increasing glutamate uptake trough modulation of BK potassium channels (Ca2 +-activated large conductance K+ channels) and phosphatidylinositol-3 kinase (PI3K) pathway. In the present study, GUO (1 mM) protected human neuroblastoma cell cultures (SH-SY5Y) from neuronal death due overproduction of reactive oxygen species (ROS) induced by coadministration of rotenone and oligomycin-A, by activating the PI3K/protein kinase B (Akt) cell signaling pathway, glycogen synthase kinase-3 beta (GSK3B]) and induction of the antioxidant enzyme heme oxygenase-1. These effects on SH-SY5Y were mediate via BK channels and the adenosine receptors A1R and A2AR. In hippocampal slices subjected to an ischemic in vitro model (OGD), GUO (100 uM) treatment prevented the excessive ROS production, mitochondrial membrane depolarization, inhibited the activation of the nuclear transcription factor NF-k B and reduced inducible Nitric oxide synthase (iNOS) levels induced by OGD. The mitogen-activated protein kinase (MAPK)/extracellular-signal regulated kinase (ERK) kinase (MEK) inhibitor, PD98059 and the A1R antagonist, DPCPX, abolished GUO induced neuroprotective effects. GUO stimulated glutamate uptake through activation of MAPK/ERK pathway, but did not involve activation of A1R. GUO also counteracted the glutamate release induced by OGD in hippocampal slices through modulation of the reverse activity of glutamate transporters. Additionally, OGD induced a decrease in glutamine synthetase activity and GUO treatment partially reversed this effect. However, GUO has not able to prevent the reduction of respiratory chain complexes activity and inhibition of oxidative phosphorylation induced OGD in rat hippocampal slices. The evaluation of GUO role on cortical astrocytic cells showed that GUO (10 uM) protects against OGD by stimulating the activity of glutamate transporters and decreasing ROS production. GUO effects on modulation of astrocytic glutamate transporters involved MAPK/ERK activation. In conclusion, GUO afford neuroprotection against ischemic and oxidative damage through the modulation of glutamatergic and purinergic systems, activation of BK potassium channels and involving the activation of PI3K and MAPK/ERK signaling pathways. Therefore, GUO is an endogenous and non-toxic nucleoside that may counteract neuroinflammation and excitotoxicity, presenting a potential neuroprotective role.