To learn why fertilization failure occurs, we seek to delineate the processes which underpin normal sperm formation. Importins are soluble proteins that selectively mediate active protein cargo transport from the cytoplasm into the nucleus. Their levels and actions control transcription, embryonic stem cell pluripotency, cellular stress adaptation, nuclear envelope assembly, and spindle formation. These highly conserved genes are essential for gamete formation in many species. We examined the distribution of four importins in adult mouse testis (C57BL/6J strain) and mature sperm, a2, a3, a4 and b3, that are encoded by genes and proteins with different expression profiles in individual germ cell populations during spermatogenesis. Using Western blots probed with commercially-supplied antibodies, these proteins were identified as present in mouse sperm (from cauda epididymal/vas deferens). These antibodies were applied in three individual experiments to fixed, paraffin-embedded testis sections and sperm captured (n=15) by Structured Illumination Microscopy (SIM) and immunofluorescent imaging. Importin subcellular localization in adult mouse testis was mapped in relationship to germ cell differentiation status and acrosome formation, with results reinforcing their potential for individual functions in sperm differentiation. We compared differences of their localization in acrosome-intact and acrosome-reacted (AR) sperm before and after the AR, which could suggest potential scaffolding and/or subcellular delivery roles. Of particular interest was the different relocation behavior between the importins aand b3 after the AR. Although all were present in the apical acrosome of the acrosome-intact sperm head, after the AR, importins a2 and a4 relocated into the sperm connecting piece, in contrast to a3, which stayed static. Further, b3 initially relocalized to equatorial segment and later covered the whole sperm head. These findings bring novel information about behavior of importins during the AR, a critical final step of sperm maturation that determines the ability of sperm to fuse with the egg.