Abstract Cellular energy production processes are composed of many Mg 2+ dependent enzymatic reactions. In fact, dysregulation of Mg 2+ homeostasis is involved in various cellular malfunctions and diseases. Recently, mitochondria, energy-producing organelles, have been known as major intracellular Mg 2+ stores. Several biological stimuli alter mitochondrial Mg 2+ concentration by intracellular redistribution. However, in living cells, whether mitochondrial Mg 2+ alteration affect cellular energy metabolism remains unclear. Mg 2+ transporter of mitochondrial inner membrane MRS2 is an essential component of mitochondrial Mg 2+ uptake system. Here, we comprehensively analyzed intracellular Mg 2+ levels and energy metabolism in Mrs2 knockdown (KD) cells using fluorescence imaging and metabolome analysis. Dysregulation of mitochondrial Mg 2+ homeostasis disrupted ATP production via shift of mitochondrial energy metabolism and morphology. Moreover, Mrs2 KD sensitized cellular tolerance against cellular stress. These results indicate regulation of mitochondrial Mg 2+ via MRS2 critically decides cellular energy status and cell vulnerability via regulation of mitochondrial Mg 2+ level in response to physiological stimuli.