Basonuclin (BNC1), a zinc finger transcriptional factor, is essential for mouse spermatogenesis. However, the regulatory mechanisms of BNC1 in spermatogenesis are poorly understood. In this study, we identified HSF2BP, a testis-specific binding protein of HSF2, as a binding partner of BNC1 by using yeast two-hybrid screening. HSF2BP could interact with and inhibit BNC1 transcriptional activity without affecting its expression level. Moreover, coexpression of HSF2BP with BNC1 resulted in a striking redistribution of BNC1 to the cytoplasm. These data suggest that HSF2BP may play a pivotal role in regulating BNC1 transcriptional activity and subcellular localization during spermatogenesis.