Propionic acidemia is caused by a deficiency of the enzyme propionyl coenzyme A carboxylase (PCC) located in the mitochondrial matrix. Cell-penetrating peptides, including transactivator of transcription (TAT), offer a potential to deliver a cargo into the mitochondrion. Here, we investigated the delivery of an α 6 β 6 PCC enzyme into mitochondria using the HIV TAT peptide at several levels: into isolated mitochondria, in patient fibroblast cells, and in a mouse model. Results from Western blots and enzyme activity assays confirmed the import of TAT-PCC into mitochondria, as well as into patient fibroblasts, where the colocalization of imported TAT-PCC and mitochondria was also confirmed by confocal fluorescence microscopy. Furthermore, a single-dose intraperitoneal injection into PCC-deficient mice decreased the propionylcarnitine/acetylcarnitine (C3/C2) ratio toward the normal level. These results show that a cell-penetrating peptide can deliver active multimeric enzyme into mitochondria in vitro , in situ , and in vivo and push the size limit of intracellular delivery achieved so far. Our results are promising for other mitochondrion-specific deficiencies.