In this study we identified Elongin B, a regulatory subunit of the trimeric elongation factor Elongin ABC, which increases the overall rate of elongation by RNA polymerase II, as a major binding partner of sperm-associated antigen 16S (SPAG16S), a component of nuclear speckles. Nuclear speckles are nuclear subcompartments involved in RNA maturation. Previously, we showed that SPAG16S is essential for spermatogenesis. In the present study, a specific antibody against mouse Elongin B was generated and reacted with a protein with the predicted size of Elongin B in the testis; immunofluorescence staining revealed that the Elongin B was located in the nuclei and residual bodies. In round spermatids, Elongin B was colocalised with splicing factor SC35 (SC35), a marker of nuclear speckles. During the first wave of spermatogenesis, Elongin B transcripts were initially detected at Postnatal Day (PND) 8, and levels were greatly increased afterwards. However, Elongin B protein was only found from PND30, when germ cells progressed through spermiogenesis. Polysomal gradient analysis of Elongin B transcripts isolated from adult mouse testes revealed that most of the Elongin B mRNA was associated with translationally inactive, non-polysomal ribonucleoproteins. An RNA electrophoretic mobility shift assay demonstrated that the 3 untranslated region of the Elongin B transcript was bound by proteins present in testis but not liver extracts. These findings suggest that post-transcriptional regulation of Elongin B occurs in the testis, which is a common phenomenon during male germ cell development. As a major binding partner of SPAG16S, Elongin B may play an important role in spermatogenesis by modulating RNA maturation.