Abstract Macrophages are important in the activation of innate immune responses and in a tissue-specific manner in the maintenance of organ homeostasis. Testicular macrophages (TM), which reside in the testicular interstitial space, comprise the largest leukocyte population in the testes and are assumed to play a relevant function in maintaining testicular immune privilege. Numerous studies have indicated that the interstitial fluid (IF) surrounding the TM has immunosuppressive properties, which may influence the phenotype of TM. However, the identity of the immunosuppressive molecules present in the IF is poorly characterized. We show that the rat testicular IF shifted GM-CSF–induced M1 toward the M2 macrophage phenotype. IF-polarized M2 macrophages mimic the properties of TM, such as increased expression of CD163, high secretion of IL-10, and low secretion of TNF-α. In addition, IF-polarized macrophages display immunoregulatory functions by inducing expansion of immunosuppressive regulatory T cells. We further found that corticosterone was the principal immunosuppressive molecule present in the IF and that the glucocorticoid receptor is needed for induction of the testis-specific phenotype of TM. In addition, TM locally produce small amounts of corticosterone, which suppresses the basal expression of inflammatory genes as a means to render TM refractory to inflammatory stimuli. Taken together, these results suggest that the corticosterone present in the testicular environment shapes the immunosuppressive function and phenotype of TM and that this steroid may play an important role in the establishment and sustenance of the immune privilege of the testis.