It has been known for a long time that mitochondria isolated from hepatocytes treated with glucagon or Ca(2+)-mobilizing agents such as phenylephrine show an increase in their adenine nucleotide (AdN) content, respiratory activity, and calcium retention capacity (CRC). Here, we have studied the role of SCaMC-3/slc25a23, the mitochondrial ATP-Mg/Pi carrier present in adult mouse liver, in the control of mitochondrial AdN levels and respiration in response to Ca(2+) signals as a candidate target of glucagon actions. With the use of SCaMC-3 knock-out (KO) mice, we have found that the carrier is responsible for the accumulation of AdNs in liver mitochondria in a strictly Ca(2+)-dependent way with an S0.5 for Ca(2+) activation of 3.3 ± 0.9 μm. Accumulation of matrix AdNs allows a SCaMC-3-dependent increase in CRC. In addition, SCaMC-3-dependent accumulation of AdNs is required to acquire a fully active state 3 respiration in AdN-depleted liver mitochondria, although further accumulation of AdNs is not followed by increases in respiration. Moreover, glucagon addition to isolated hepatocytes increases oligomycin-sensitive oxygen consumption and maximal respiratory rates in cells derived from wild type, but not SCaMC-3-KO mice and glucagon administration in vivo results in an increase in AdN content, state 3 respiration and CRC in liver mitochondria in wild type but not in SCaMC-3-KO mice. These results show that SCaMC-3 is required for the increase in oxidative phosphorylation observed in liver mitochondria in response to glucagon and Ca(2+)-mobilizing agents, possibly by allowing a Ca(2+)-dependent accumulation of mitochondrial AdNs and matrix Ca(2+), events permissive for other glucagon actions.