Interactions between endoplasmic reticulum (ER) and mitochondria are key components of essential cellular functions. Indeed, these membrane appositions are necessary for proper Ca2+ transfer from ER to mitochondria, to regulate lipid metabolism, apoptosis, and inflammation. We report that the ER protein WFS1 interacts with the neuronal calcium sensor protein NCS1 to regulate mitochondria associated-ER membrane formation. Mutations in the WFS1 gene are associated with Wolfram syndrome, a rare neurodegenerative disease. We demonstrated that human WFS1-deficient cells lack NCS1 and fail to tether ER and mitochondria, resulting in a decrease in Ca2+ transfer and mitochondrial respiration. Interestingly, we showed that NCS1 overexpression in WFS1-deficient cells restored ER–mitochondria interactions and calcium exchange. Our results suggest that WFS1 regulates ER tethering to mitochondria through NCS1 and that restoration of NCS1 expression could be a therapeutic tool for restoring calcium signaling at the mitochondria associated-ER membrane interface and mitochondrial function in Wolfram syndrome.