Amyotrophic lateral sclerosis (ALS) is an adult onset progressive motor neuron disease with no cure. Transgenic mice overexpressing familial ALS associated human mutant SOD1 are a commonly used model for examining disease mechanisms. Presently, it is well accepted that alterations in motor neuron excitability and spinal circuits are pathological hallmarks of ALS, but the underlying molecular mechanisms remain unresolved. Here, we sought to understand whether the expression of mutant SOD1 protein could contribute to altering processes governing motor neuron excitability. We used the conformation specific antibody B8H10 which recognizes a misfolded state of SOD1 (misfSOD1) to longitudinally identify its interactome during early disease stage in SOD1G93A mice. This strategy identified a direct isozyme-specific association of misfSOD1 with Na(+)/K(+)ATPase-α3 leading to the premature impairment of its ATPase activity. Pharmacological inhibition of Na(+)/K(+)ATPase-α3 altered glutamate receptor 2 expression, modified cholinergic inputs and accelerated disease pathology. After mapping the site of direct association of misfSOD1 with Na(+)/K(+)ATPase-α3 onto a 10 amino acid stretch that is unique to Na(+)/K(+)ATPase-α3 but not found in the closely related Na(+)/K(+)ATPase-α1 isozyme, we generated a misfSOD1 binding deficient, but fully functional Na(+)/K(+)ATPase-α3 pump. Adeno associated virus (AAV)-mediated expression of this chimeric Na(+)/K(+)ATPase-α3 restored Na(+)/K(+)ATPase-α3 activity in the spinal cord, delayed pathological alterations and prolonged survival of SOD1G93A mice. Additionally, altered Na(+)/K(+)ATPase-α3 expression was observed in the spinal cord of individuals with sporadic and familial ALS. A fraction of sporadic ALS cases also presented B8H10 positive misfSOD1 immunoreactivity, suggesting that similar mechanism might contribute to the pathology.