Spermatogenesis is an important developmental process requiring stage-specific expression of genes highly dependent on regulated nuclear protein import. BRCA1 binding protein 2 (BRAP2), a negative regulator of nuclear import (NRNI) of proteins implicated in key cellular processes such as viral infection, cell cycle and cancer progression is highly expressed in testis. To identify potential binding partners of BRAP2 in spermatogenesis, a yeast-2-hybrid screen was performed on an adult human testis library, with various proteins of interest, including the PH domain and leucine rich repeat protein phosphatase 1 (PHLPP1), A-Kinase anchor protein (AKAP3) and DNA methyl transferase 1 (DNMT1) were identified as novel candidates. Interactions were confirmed by coimmunoprecipitation assays using lysates from adult mouse testis. Immunohistochemistry revealed the presence of BRAP2 in spermatocytes, round spermatids and elongated spermatids, concomitant with the cytoplasmic expression of BRAP2 binding partners in these cell types. BRAP2 and binding partners were also detected on sperm by indirect immunofluorescence. Ectopic expression of truncated derivatives of BRAP2 was found to significantly reduce nuclear localisation of binding partners in transfected cells, indicating the capability of BRAP2 to act as their cytoplasmic retention factor. Importantly amino acid residues 442-570 of BRAP2 showed the highest NRNI activity with maximal reduction in nuclear accumulation of PHLPP1, AKAP3 and DNMT1. We propose that BRAP2 may regulate the nuclear import of specific binding partners critical for normal spermatogenesis/testicular development.