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.