Mitochondria-associated ER membranes (MAM) are transient functional domains in the endoplasmic reticulum (ER) in close apposition to mitochondria involved in multiple metabolic functions, including the regulation of mitochondria functionality. Specifically, MAM interactions with mitochondria contribute to the regulation of mitochondrial dynamics, calcium transference between both organelles and the composition of mitochondrial membranes. In addition, recent data indicate that alterations in MAM-mitochondria contacts are associated with impairments in glucose metabolism and insulin resistance phenotypes, but the mechanism behind these defects is unknown. Human embryonic stem cell (ESC)-derived motor neurons (hMNs) and mouse models with pathogenic mutations in superoxide dismutase 1 (SOD1) have been shown to present with a progressive disruption of MAM structure and function, as it occurs in other models of amyotrophic lateral sclerosis (ALS). In this work, we have found that impairments in the activation of MAM in the context of SOD1 mutations, hinder the use of glucose-derived pyruvate as a mitochondrial fuel and trigger a shift in mitochondrial substrates from pyruvate to fatty acids. We also show that, over time, this change in mitochondrial fuels induces significant alterations in mitochondrial electron flow and in the active/de-active (A/D) status of complex I in mutant hMNs and spinal cord tissues, but not in brain. Our data agree with a role for MAM in the maintenance and regulation of cellular glucose metabolism and the selection of mitochondrial substrates and suggest that MAM disruption in ALS could be the underlying cause of the bioenergetic deficits observed in the disease.