Mitochondria play an important role in cell life and in the regulation of cell death. In addition, mitochondrial dysfunction contributes to a wide range of neuropathologies. The nucleoside Guanosine (GUO) is an endogenous molecule, presenting antioxidant properties, possibly due to its direct scavenging ability and/or from its capacity to activate the antioxidant defense system. GUO demonstrate a neuroprotective effect due to the modulation of the glutamatergic system and maintenance of the redox system. Thus, considering the few studies focused on the direct effects of GUO on mitochondrial bioenergetics, we designed a study to evaluate the in vitro effects of GUO on rat mitochondrial function, as well as against Ca²⁺-induced impairment. Our results indicate that GUO prevented mitochondrial dysfunction induced by Ca²⁺ misbalance, once GUO was able to reduce mitochondrial swelling in the presence of Ca²⁺, as well as ROS production and hydrogen peroxide levels, and to increase manganese superoxide dismutase activity, oxidative phosphorylation and tricarboxylic acid cycle activities. Our study indicates for the first time that GUO could direct prevent the mitochondrial damage induced by Ca²⁺ and that these effects were not related to its scavenging properties. Our data indicates that GUO could be included as a new pharmacological strategy for diseases linked to mitochondrial dysfunction.