The endoplasmic reticulum (ER) was long considered to be the only compartment of the eukaryotic cell in which protein folding is accompanied by enzyme-catalyzed disulfide bond formation. However, it has recently become evident that cells harbor a second oxidizing compartment, the mitochondrial intermembrane space, where disulfide formation facilitates protein translocation from the cytosol. Moreover, protein oxidation has been implicated in many mitochondria-associated processes central for human health such as apoptosis, aging, and regulation of the respiratory chain. Whereas the machineries of ER and mitochondria both form disulfides between cysteine residues, they do not share evolutionary origins and exhibit distinct mechanistic properties. Here, we summarize the current knowledge of these oxidation systems and discuss their functional similarities and differences.