Description: Objective— We explored the role of endoplasmic reticulum (ER)–mitochondria Ca 2+ cross talk involving voltage-dependent anion channel-1 (VDAC1)/glucose-regulated protein 75/inositol 1,4,5-trisphosphate receptor 1 complex and mitofusin 2 in endothelial cells during hypoxia/reoxygenation (H/R), and investigated the protective effects of acetylcholine. Approach and Results— Acetylcholine treatment during reoxygenation prevented intracellular and mitochondrial Ca 2+ increases and alleviated ER Ca 2+ depletion during H/R in human umbilical vein endothelial cells. Consequently, acetylcholine enhanced mitochondrial membrane potential and inhibited proapoptotic cascades, thereby reducing cell death and preserving endothelial ultrastructure. This effect was likely mediated by the type-3 muscarinic acetylcholine receptor and the phosphatidylinositol 3-kinase/Akt pathway. In addition, interactions among members of the VDAC1/glucose-regulated protein 75/inositol 1,4,5-trisphosphate receptor 1 complex were increased after H/R and were associated with mitochondrial Ca 2+ overload and cell death. Inhibition of the partner of the Ca 2+ channeling complex (VDAC1 siRNA) or a reduction in ER–mitochondria tethering (mitofusin 2 siRNA) prevented the increased protein interaction within the complex and reduced mitochondrial Ca 2+ accumulation and subsequent endothelial cell death after H/R. Intriguingly, acetylcholine could modulate ER–mitochondria Ca 2+ cross talk by inhibiting the VDAC1/glucose-regulated protein 75/inositol 1,4,5-trisphosphate receptor 1 complex and mitofusin 2 expression. Phosphatidylinositol 3-kinase siRNA diminished acetylcholine-mediated inhibition of mitochondrial Ca 2+ overload and VDAC1/glucose-regulated protein 75/inositol 1,4,5-trisphosphate receptor 1 complex formation induced by H/R. Conclusions— Our data suggest that ER–mitochondria interplay plays an important role in reperfusion injury in the endothelium and may be a novel molecular target for endothelial protection. Acetylcholine attenuates both intracellular and mitochondrial Ca 2+ overload and protects endothelial cells from H/R injury, presumably by disrupting the ER–mitochondria interaction.