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We explored the role played by AKAP121 in the regulation of mitochondrial membrane potential and mitochondrial calcium concentration ([Ca2+]m), as indices of mitochondrial functions. We found that AKAP121 overexpression hyperpolarizes inner mitochondrial membrane in a PKA and PTPD1 dependent manner. Cells transiently transfected with the AKAP121PKAm, that does not bind PKA, or AKAP121 45-110, that does not bind PTPD1/src, displayed depolarization of inner mitochondrial membrane, thus suggesting the role of PKA and src in the control of mitochondrial oxidative phosphorilation. In addition, the overexpression of AKAP121 significantly decreased [Ca2+]m, whereas cells transfected with AKAP121 45-110, or with AKAP121PKAm, displayed an increase of [Ca2+]m. Moreover, transiently or stably transfected AKAP121 exposed to a hypoxic insults showed a marked impairment of mitochondrial membrane depolarization compared with wild type cells. These results demonstrate that AKAP121, by integrating PKA and src signalling to mitochondria, controls oxidative phosphorylation and mitochondrial calcium homeostasis.