Thiol groups of cysteine residues represent redox centers involved in multiple biological functions. It has been postulated that changes in the redox status of mammalian epididymal spermatozoa contribute to the sperm maturation process. The present work shows the thiol-disulfide protein profile of stallion epididymal spermatozoa achieved by two-dimension electrophoresis and MALDI-TOF/TOF mass spectrometry of proteins labeled with a thiol-reactive fluorescent tag, monobromobimane. Our results have shown the formation of disulfide bonds in several sperm protein fractions during the epididymal maturation process. The majority of the oxidized thiol sperm proteins identified correspond to structural molecules of the flagellum (as the outer dense fiber-1 protein – ODF1), followed by glycolytic enzymes (as glyceraldehyde-3-phosphate dehydrogenase spermatogenic), antioxidant protectors (as glutathione S-transferase and phospholipid hydroperoxide glutathione peroxidase – PHGPx). The magnitude of the thiol oxidation differs between proteins, and was more drastic in polypeptides with molecular weights of up to 33kDa, identified as ODF1 and PHGPx. A kinase anchor protein, a voltage-dependent anion channel protein and a zona pellucida-binding protein were also found in the polypeptide samples that contained oxidized SH groups. These proteins may be modified or controlled by the mechanisms involved in the cysteine-redox changes, corroborating the belief that a correct degree of protein oxidation is required for the stabilization of sperm structure, protection against oxidative damage, induction of progressive sperm motility and fertilization.