Glucocorticoids (GCs) as mediators of the stress response may affect Leydig cell function by inhibiting either luteinizing hormone receptor expression or testosterone biosynthesis. The isozymes 11β-hydroxysteroid dehydrogenase (11βHSD) 1 and 11βHSD2 control the intracellular cortisol levels. Little is known about the effects of stress on fertility in the equine. The objective of the present study was to determine the presence and cellular localization of glucocorticoid receptors (GCR) and glucocorticoid-metabolizing enzymes (11βHSD1 and 11βHSD2) in equine epididymal and testicular tissue with special regard to sexual maturation. Testicular and epididymal tissue was collected from 21 healthy stallions, and four age groups were designed: pre-pubertal, young, mature and older horses. Immunohistochemistry (IHC) analysis and quantitative real-time PCR (qRT-PCR) were used. Pre-pubertal horses showed higher testicular gene expression of 11βHSD1, 11βHSD2 and GCR than horses of all other groups (p < 0.05). A positive intranuclear immunoreaction for GCR was seen in epithelial cells of caput, corpus and cauda epididymidis and in Leydig cells. Significant differences (p < 0.05) between age groups occurred. The number of Leydig cells staining positive for GCR was highest in immature stallions (p < 0.05). The enzyme 11βHSD1 was localized in epithelial cells of the caput and corpus epididymidis and in Leydig cells. As determined by enzyme assay, nicotinamide adenine dinucleotide (NAD)-dependant dehydrogenase (oxidation) activity was not detected in testicular tissue from immature stallions but in all other age groups (n = 3 per group). Results of this study suggest a contribution of GCs to maturation of male reproductive tissue in horses. In mature stallions, expression of 11βHSD enzymes and the oxidative 11βHSD activity in Leydig cells and epididymal basal and principal cells suggest a protective role on these tissues contributing to physiological intracellular glucocorticoid concentrations.