The organization and mutual interactions between endoplasmic reticulum (ER) and mitochondria modulate key aspects of cell pathophysiology. Several proteins have been suggested to be involved in keeping ER and mitochondria at a correct distance. Among them, in mammalian cells, mitofusin 2 (Mfn2), located on both the outer mitochondrial membrane and the ER surface, has been proposed to be a physical tether between the two organelles, forming homotypic interactions and heterocomplexes with its homolog Mfn1. Recently, this widely accepted model has been challenged using quantitative EM analysis. Using a multiplicity of morphological, biochemical, functional, and genetic approaches, we demonstrate that Mfn2 ablation increases the structural and functional ER–mitochondria coupling. In particular, we show that in different cell types Mfn2 ablation or silencing increases the close contacts between the two organelles and strengthens the efficacy of inositol trisphosphate (IP3)-induced Ca²⁺ transfer from the ER to mitochondria, sensitizing cells to a mitochondrial Ca²⁺ overload-dependent death. We also show that the previously reported discrepancy between electron and fluorescence microscopy data on ER–mitochondria proximity in Mfn2-ablated cells is only apparent. By using a different type of morphological analysis of fluorescent images that takes into account (and corrects for) the gross modifications in mitochondrial shape resulting from Mfn2 ablation, we demonstrate that an increased proximity between the organelles is also observed by confocal microscopy when Mfn2 levels are reduced. Based on these results, we propose a new model for ER–mitochondria juxtaposition in which Mfn2 works as a tethering antagonist preventing an excessive, potentially toxic, proximity between the two organelles.