The difference between transcriptional and translational activities of genes is one of the remarkable features in spermatogenesis. In this study, we characterized MIF4GD (MIF4G domain containing), a possible translational regulator, to uncover the function during rat spermatogenesis. Western blot analysis revealed the presence of two forms of MIF4GD with molecular sizes of 25 and 16 kDa in various tissues including the testis. An additional 20-kDa form of MIF4GD was also found exclusively in the testis. These three proteins were abundantly present in pachytene spermatocytes and haploid spermatids. Importantly, the 16-kDa MIF4GD was distinguished from the 25- and 20-kDa proteins by presence in fractions of 40S subunits and ribosomes of spermatogenic cells according to analysis of subcellular localizations. These results suggest that the 16-kDa MIF4GD may function as a translational regulator in spermiogenesis.