Balance of spermatogonial stem and progenitor cell (SSPC) self-renewal and differentiation is essential for the homeostasis of spermatogenesis and the maintenance of male fertility. Regulation of SSPC function requires a complex interplay of intrinsic and extrinsic niche-derived factors. In this study, we identified the splicing factor RBM5 as a novel regulator of spermatogonia differentiation. Male mice carrying an ENU-induced missense mutation (R263P) in the second RNA recognition motif (RRM2) of RBM5 were sterile due to a round spermatid arrest, which ultimately led to azoospermia. We have shown that RBM5 is an essential splicing factor in round spermatids and the R263P mutation resulted in aberrant splicing in several target pre-mRNAs that are required for spermatid differentiation. Within the adult mouse testis, RBM5 localises to the nucleus of somatic and germ cells including spermatogonia, spermatocytes and round spermatids. Further a stereological analysis revealed that in addition to the spermatid arrest phenotype Rbm5 mutant mice have a decreased conversion of spermatogonia into spermatocytes and significant loss of late spermatocytes. In order to investigate defects during the transition from spermatogonia to spermatocytes, Rmb5 mutant versus wild type day 3 and 7 postnatal testes were stained for PLZF as a marker for undifferentiated spermatogonia. The number of undifferentiated spermatogonia (PLZF+ cells) per tubule observed in postnatal day 3 in the Rbm5 mutant testes was normal, however, a significant reduction compared to that in wild type animals was seen at postnatal day 7, suggesting a failure of spermatogonial commitment. Consistently, FACS analyses of the adult testes showed a significant increase in number of undifferentiated spermatogonia (PLZF+/c-Kit-/Ki67-) in the mutant compared to wild-type testes. Taken together, our findings define for the first time a critical role for RBM5 in spermatogonia differentiation.