The endogenously produced gasotransmitter carbon monoxide (CO) has been studied as a factor involved in cytoprotection, homeostasis and anti-inflammation1. Several evidences show CO targeting mitochondria and small amounts of reactive oxygen species (ROS) are described as signaling factors in CO’s biological mode of action. Mitochondria are the main source of ROS and are also key organelles in orchestrating cell function: metabolism, cell death control and redox signaling2. Mitochondria can act as calcium buffering organelles and respond to it by two distinct ways. Calcium entry in mitochondrial matrix stimulates ATP production by improving mitochondrial respiration and increasing pyruvate dehydrogenase activity3. Nevertheless, when the capacity of mitochondria is exceeded, calcium overload promotes the opening of permeability transition pore (PTP), which is a high-conductance channel, leading to mitochondrial depolarization, permeabilization and ultimately cell death3. Astrocytes are most abundant glial cells and essential for neuronal function, namely metabolic and physical support, expression of neurotransmitters and promotion of neuroprotection. Primary culture of astrocytes is the cellular model. Non-synaptic mitochondria isolated from rodent cortex are the cell-free system used for studying the direct effect of CO on mitochondria. Two CO sources are used: CO gas through PBS saturated solutions and CO-releasing molecule -A1 (CORM-A1). Low concentrations of CO partially inhibited oxidative stress-induced apoptosis in astrocytes, by preventing caspase-3 activation, mitochondrial potential depolarization and plasmatic membrane permeability. Furthermore, CO directly targets non-synaptic mitochondria and inhibits PTP opening by partially inhibiting (i) loss of potential, (ii) the opening of a non specific pore through the inner membrane and (iii) mitochondrial swelling, which are induced by calcium or atractyloside (a ligand of adenine nucleotide translocator, ANT) 4,5. Thus, CO limits the release of cytochrome c from mitochondria into the cytosol, which limited caspase activation and triggering of apoptotic cascade in astrocytes. CO induced ROS generation, and their scavenging by β-carotene, decreased CO cytoprotection in intact cells, as well as in isolated mitochondria, revealing the key role of ROS. Additionally, CO promotes a slight increase on mitochondrial oxidized glutathione, which in turn modified ANT protein by glutathionylation. Glutathionylation of ANT increased the ATP/ADP exchange through mitochondrial inner membrane and limited the opening of PTP4. In addition, CO exposure enhanced ATP generation, which was accompanied by an increase on specific oxygen consumption, a decrease on lactate production and a reduction of glucose use, indicating an improvement of oxidative phosphorylation. Accordingly, CO increased cytochrome c oxidase specific enzymatic activity and enhanced mitochondrial population. The CO-induced oxidative metabolic improvement is dependent on Bcl-2 expression, since silencing Bcl-2 expression with siRNA reverted cytoprotection and metabolic improvement6. Dysfunctional mitochondrial can be eliminated by mitophagy, which is a crucial process for maintaining their good function and quality control7. In astrocytes, CO promotes mitophagy at 1h of treatment, while following 24h mitochondrial population is back to basal levels, indicating that CO contributes to mitochondrial turnover. Furthermore, CO limits astrocytic cell death in an autophagic dependent manner. All these data suggest that CO modulates calcium entry into mitochondria. Thus, CO prevents astrocytic cell death and improves cell metabolism by targeting mitochondria, and some of the underlying molecular mechanism are disclosed.