The disulfide relay system in the intermembrane space of mitochondria is of crucial importance for mitochondrial biogenesis. Major players in this pathway are the oxidoreductase Mia40 that oxidizes substrates and the sulfhydryl oxidase Erv1 that reoxidizes Mia40. To analyze in detail the mechanism of this oxidative pathway and the interplay of its components, we reconstituted the complete process in vitro using purified cytochrome c, Erv1, Mia40, and Cox19. Here, we demonstrate that Erv1 dimerizes noncovalently and that the subunits of this homodimer cooperate in intersubunit electron exchange. Moreover, we show that Mia40 promotes complete oxidation of the substrate Cox19. The efficient formation of disulfide bonds is hampered by the formation of long-lived, partially oxidized intermediates. The generation of these side products is efficiently counteracted by reduced glutathione. Thus, our findings suggest a role for a glutathione-dependent proofreading during oxidative protein folding by the mitochondrial disulfide relay.