The potent lipid mediator sphingosine-1-phosphate (S1P) regulates diverse physiological processes by binding to 5 specific GPCRs, although it also has intracellular targets. Here, we demonstrate that S1P, produced in the mitochondria mainly by sphingosine kinase 2 (SphK2), binds with high affinity and specificity to prohibitin 2 (PHB2), a highly conserved protein that regulates mitochondrial assembly and function. In contrast, S1P did not bind to the closely related protein PHB1, which forms large, multimeric complexes with PHB2. In mitochondria from SphK2-null mice, a new aberrant band of cytochrome-c oxidase was detected by blue native PAGE, and interaction between subunit IV of cytochrome-c oxidase and PHB2 was greatly reduced. Moreover, depletion of SphK2 or PHB2 led to a dysfunction in mitochondrial respiration through cytochrome-c oxidase. Our data point to a new action of S1P in mitochondria and suggest that interaction of S1P with homomeric PHB2 is important for cytochrome-c oxidase assembly and mitochondrial respiration.—Strub, GM, Paillard, M., Liang, J., Gomez, L., Allegood, JC, Hait, NC, Maceyka, M., Price, MM, Chen, Q., Simpson, DC, Kordula, T., Milstien, S., Lesnefsky, EJ, Spiegel, S. Sphingosine-1-phosphate produced by sphingosine kinase 2 in mitochondria interacts with prohibitin 2 to regulate complex IV assembly and respiration.