Macrophage migration inhibitory factor (MIF) is a multifunctional molecule. MIF was originally identified as a T‐cell‐derived factor responsible for the inhibition of macrophage migration. In testicular tissue of adult rats, MIF is constitutively expressed by Leydig cells under physiological conditions. The aim of this study was to examine MIF levels in testicular homogenates from different aged mice, and the capacity of Sertoli cells to produce it. We also examined MIF involvement in spermatogonial cell migration. Similar levels of MIF protein were detected in testicular homogenates of mice of different ages (1–8‐week‐old). However, the RNA expression levels of MIF were high in 1‐week‐old mice and significantly decreased with age compared to 1‐week‐old mice. MIF was stained in Sertoli, Leydig cells, and developed germ cells in the seminiferous tubules. Isolated Sertoli cells from 1‐week‐old mice stained to MIF. Cultures of Sertoli cells from 1‐week‐old mice produced and expressed high levels of MIF which significantly decreased with age. MIF was localized in the cytoplasm and nucleus of Sertoli cell cultures isolated from 1‐week‐old mice; however, it was localized only in the cytoplasm and branches of cultures isolated from 8‐week‐old mice. MIFR was detected in GFRα1 and Sertoli cells. MIF could induce migration of spermatogonial cells, and this effect was synergistic with glial cell‐line neurotrophic factor. Our results show, for the first time, the capacity of Sertoli cells to produce MIF under normal conditions and that MIFR expressed in GFRα1 and Sertoli cells. We also showed that MIF induced spermatogonial cell migration. J. Cell. Physiol. 232: 2869–2877, 2017. © 2016 Wiley Periodicals, Inc. Our study showed that Sertoli cells and spermatogonial cells generate MIF and express MIF receptor. MIF could induce spermatogonial cell migration.