We tested the hypothesis that sheep breed can influence the sperm quality and seminal plasma (SP) composition and investigated any potential relationship between SP proteins and antioxidant enzyme activities (AO) with sperm quality. Ejaculates from twelve rams of three breeds were obtained during the rainy season at high altitudes, and sperm quality was automatically evaluated (CASA-Hamilton Thorne). The AO of superoxide dismutase, glutathione peroxide and glutathione reductase (GR) in SP was evaluated and total proteins were separated by SDS-PAGE. Comparative analyses of semen quality parameters between breeds revealed that Creole and Hampshire breeds had a higher sperm quality compared with Romney Marsh (p<0.05), although no difference in AO was found. GR activity was negatively correlated (p<0.05) with several kinematic variables and positively (p<0.05) with morphological abnormalities. The highest SP protein concentration was found in semen collected from Hampshire males compared with that from Creole and Romney Marsh (p<0.05). SDS-PAGE analysis showed the presence of 32 protein bands in SP with molecular weights between 334 and 10 kDa. Differences (p<0.05) between breeds in bands of 43, 25, 22 and 20 kDa were observed. These results evidence a relationship between the protective effect of the antioxidant enzyme system in SP of three ram types under high altitude and tropical conditions and semen quality. Our findings also suggest that the identified proteins might play an important role in sperm physiology and quality.