Proper biogenesis of mitochondria is a crucial process for cell viability. The majority of mitochondrial proteins are synthesized on cytosolic ribosomes, therefore they have to be selectively transported to the final destination site in mitochondria. Mitochondria developed several machineries specialized in recognition and sorting of precursor proteins. The MIA (Mitochondrial Intermembrane Space Assembly) pathway is generally viewed to be dedicated to the redox-dependent import and biogenesis of intermembrane space (IMS) proteins. The Mia40 oxidoreductase, a central component of the pathway, is responsible for  the  maturation of the incoming precursor proteins. This process involves the transfer of disulfide bonds from Mia40 to the precursors proteins followed by their oxidative folding. Our results indicate that Mia40 is also involved in the biogenesis of the proteins localized in other mitochondrial compartments.We present the first evidence that the function of Mia40 is not restricted to the transport and oxidative folding of soluble IMS precursor proteins. We show that Mia40 is directly involved in the biogenesis of the inner membrane protein, Tim22 – an essential core component of the TIM22 translocase. This highly conserved membrane protein forms a disulfide-bonded intermediate with Mia40 upon import into isolated mitochondria. Interestingly, Mia40 recognizes and binds Tim22 precursor also via non-covalent interactions. We propose that Mia40 is not only responsible for disulfide-bond formation, but also serves as a chaperone that assists the Tim22 protein in its integration into the inner membrane of mitochondria.