The crosstalk between different organelles allows for the exchange of proteins, lipids and ions. Endoplasmic reticulum (ER) and mitochondria are physically linked and signal through the mitochondria-associated membrane (MAM) to regulate the transfer of Ca2+ from ER stores into the mitochondrial matrix, thereby affecting mitochondrial function and intracellular Ca2+ homeostasis. The chaperone glucose-regulated protein 75 (GRP75) is a key protein expressed at the MAM interface which regulates ER-mitochondrial Ca2+ transfer. Previous studies revealed that modulation of GRP75 expression largely affected mitochondrial integrity and vulnerability to cell death. In the present study, we show that genetic ablation of GRP75, by weakening ER-mitochondrial junctions, provided protection against mitochondrial dysfunction and cell death in a model of glutamate-induced oxidative stress. Interestingly, GRP75 silencing attenuated both cytosolic and mitochondrial Ca2+ overload in conditions of oxidative stress, blocked the formation of reactive oxygen species and preserved mitochondrial respiration. These data revealed a major role for GRP75 in regulating mitochondrial function, Ca2+ and redox homeostasis. In line, GRP75 overexpression enhanced oxidative cell death induced by glutamate. Overall, our findings suggest weakening ER-mitochondrial connectivity by GRP75 inhibition as a novel protective approach in paradigms of oxidative stress in neuronal cells.