Mitochondrial Ca2+ signals have been proposed to accelerate oxidative metabolism and ATP production to match Ca2+-activated energy-consuming processes. Efforts to understand the signaling role of mitochondrial Ca2+ have been hampered by the inability to manipulate matrix Ca2+ without directly altering cytosolic Ca2+. We were able to selectively buffer mitochondrial Ca2+ rises by targeting the Ca2+-binding protein SING to the matrix. We find that matrix Ca2+ controls signal-dependent NAD(P)H formation, respiration, and ATP changes in intact cells. Furthermore, we demonstrate that matrix Ca2+ increases are necessary for the amplification of sustained glucose-dependent insulin secretion in cells. Through the regulation of NAD(P)H in adrenal glomerulosa cells, matrix Ca2+ also acts as a positive signal in reductive biosynthesis, which stimulates aldosterone secretion. Our dissection of cytosolic and mitochondrial Ca2+ signals reveals the physiological importance of matrix Ca2+ in energy metabolism required for signal-dependent hormone secretion.