TDP-43 is an evolutionarily conserved ubiquitously expressed DNA/RNA-binding protein. Although recent studies have shown its association with a variety of neurodegenerative disorders, the function of TDP-43 remains poorly understood. Here we address TDP-43 function using spermatogenesis as a model system. We previously showed that TDP-43 binds to the testis-specific mouse acrv1 gene promoter in vitro via two GTGTGT-motifs and that mutation of these motifs led to premature transcription in spermatocytes of an otherwise round spermatid-specific promoter. The present study tested the hypothesis that TDP-43 represses acrv1 gene transcription in spermatocytes. Plasmid chromatin immunoprecipitation demonstrated that TDP-43 binds to the acrv1 promoter through GTGTGT motifs in vivo. Reporter gene assays showed that TDP-43 represses acrv1 core promoter-driven transcription via the N-terminal RRM1 domain in a histone deacetylase-independent manner. Consistent with repressor role, ChIP on physiologically isolated germ cells confirmed that TDP-43 occupies the endogenous acrv1 promoter in spermatocytes. Surprisingly, however, TDP-43 remains at the promoter in round spermatids, which express acrv1 mRNA. We show that RNA binding-defective TDP-43, but not splice variant isoforms, relieve repressor function. Transitioning from repressive to active histone marks has little effect on TDP-43 occupancy. Finally, we found that RNA polymerase II is recruited but paused at the acrv1 promoter in spermatocytes. Because mutation of TDP-43 sites caused premature transcription in spermatocytes in vivo, TDP-43 may be involved in pausing RNAPII at the acrv1 promoter in spermatocytes. Overall, our study shows that TDP-43 is a transcriptional repressor and that it regulates spatiotemporal expression of the acrv1 gene during spermatogenesis.