Gα12 constitutes, along with Gα13, one of the four families of α subunits of heterotrimeric G proteins. We found that the N terminus of Gα12, but not those of other Gα subunits, contains a predicted mitochondrial targeting sequence. Using confocal microscopy and cell fractionation, we demonstrated that up to 40% of endogenous Gα12 in human umbilical vein endothelial cells colocalize with mitochondrial markers. N-terminal sequence of Gα12 fused to GFP efficiently targeted the fusion protein to mitochondria. Gα12 with mutated mitochondrial targeting sequence was still located in mitochondria, suggesting the existence of additional mechanisms for mitochondrial localization. Lysophosphatidic acid, one of the known stimuli transduced by Gα12/13, inhibited mitochondrial motility, while depletion of endogenous Gα12 increased mitochondrial motility. Gα12Q229L variants uncoupled from RhoGEFs (but not fully functional activated Gα12Q229L) induced transformation of the mitochondrial network into punctate mitochondria and resulted in a loss of mitochondrial membrane potential. All examined Gα12Q229L variants reduced phosphorylation of Bcl-2 at Ser-70, while only mutants unable to bind RhoGEFs also decreased cellular levels of Bcl-2. These Gα12 mutants were also more efficient Hsp90 interactors. These findings are the first demonstration of a heterotrimeric G protein α subunit specifically targeted to mitochondria and involved in the control of mitochondrial morphology and dynamics.—Andreeva, AV, Kutuzov, MA, Voyno-Yasenetskaya, TA Gα12 is targeted to the mitochondria and affects mitochondrial morphology and motility.