Abstract Spermatozoa are redox-regulated cells, and stallion spermatozoa, in particular, present an intense mitochondrial activity in which large amounts of reactive oxygen species (ROS) are produced. To maintain the redox potential under physiological conditions, sophisticated mechanisms ought to be present, particularly in the mitochondria. In the present study, we investigated the role of the SLC7A11 antiporter. This antiporter exchanges intracellular glutamate for extracellular cystine. In the spermatozoa, cystine is reduced to cysteine and used for GSH synthesis. The importance of the antiporter for mitochondrial functionality was studied using flow cytometry and UHPLC/MS/MS approaches. Intracellular GSH increased in the presence of cystine, but was reduced in the presence of Buthionine sulphoximine (BSO), a γ-glutamylcysteine synthetase inhibitor (P < 0.001). Inhibition of the SLC7A11 antiporter with sulfasalazine caused a dramatic drop in intracellular GSH (P < 0.001) and in the percentage of spermatozoa showing active mitochondria (P < 0.001). These findings suggest that proper functionality of this antiporter is required for the mitochondrial function of spermatozoa. We also describe that under some conditions, glutamate may be metabolized following non-conventional pathways, also contributing to sperm functionality. We provide evidences, that the stallion spermatozoa have important metabolic plasticity, and also of the relation between redox regulation and metabolic regulation. These findings may have important implications for the understanding of sperm biology and the development of new strategies for sperm conservation and treatment of male factor infertility.