Motoneurons (MNs) are particularly affected by inhibition of mitochondrial metabolism, which has been linked to their selective vulnerability during pathophysiological states like hypoxia and amyotrophic lateral sclerosis, a fatal neurodegenerative disorder. To elucidate underlying events, we utilized sodium cyanide (CN) as a pharmacological inhibitor of complex IV of the mitochondrial respiratory chain ("chemical hypoxia") and investigated the cellular response in vulnerable and resistant MN types. Bath application of 2mM CN activated TTX-insensitive Na⁺ conductances in vulnerable hypoglossal MNs, which depolarised MNs by 10.2 ± 1.1 mV and increased their action potential activity. This response was mimicked by sodium azide (2mM) and largely prevented by pre-incubation with the antioxidants ascorbic acid (1mM) and trolox (750µM), indicating an involvement of reactive oxygen species (ROS) in the activation mechanism. CN also elevated cytosolic [Ca²⁺] levels through i) Ca²⁺ release from mitochondria-controlled stores, ii) significant retardation of cytosolic Ca²⁺ clearance rates, even when cytosolic ATP levels were held constant during whole-cell recording and iii) secondary Ca²⁺ influx during elevated firing rates. Blocking mitochondrial ATP production additionally raised cytosolic Ca²⁺ levels and prolonged recovery of Ca²⁺ transients with a delay of 5- 6 minutes. Comparative studies on hypoglossal MNs, facial MNs and dorsal vagal neurons suggested that CN- responses were dominated by activation of K⁺ conductances in resistant motoneurons, thus reducing the excitability during mitochondrial inhibition. In summary, our observations therefore support a model where selective MN vulnerability results from a synergistic accumulation of risk factors, including low cytosolic Ca²⁺ buffering, strong mitochondrial impact on [Ca²⁺]i, and a mitochondria-controlled increase in electrical excitability during metabolic disturbances.