RNA‐binding proteins (RBP) are important facilitators of post‐transcriptional gene regulation. We have previously established that nuclear overexpression of the RBP Musashi‐2 (MSI2) during male germ cell maturation is detrimental to sperm cell development and fertility. Herein we determine the genes and pathways impacted by the upregulation of Msi2. Microarray analysis and qPCR confirmed differential gene expression in factors fundamental to the cell cycle, cellular proliferation, and cell death. Similarly, comparative protein expression analysis via iTRAQ, immunoblot, and immunolocalization, identified differential expression and localization of important regulators of transcription, translation, RNA processing, and spermatogenesis. Specifically, the testis‐expressed transcription factor, Tbx1, and the piRNA regulator of gamete development, Piwil1, were both found to be targeted for translational repression by MSI2. This study provides key evidence to support a fundamental role for MSI2 in post‐transcriptional regulation during male gamete development. In this study, we have utilized comparative gene and protein expression analyses to determine the impact of Msi2 overexpression on testicular germ cell development and, identify a potential mechanism for the action of MSI2 in post‐transcriptional regulation. This detailed analysis uncovered two testis‐specific MSI2‐RNA binding targets in transcription factor Tbx1 and piRNA regulator Piwil1, with evidence for their translational repression in spermatids.