Spermatogenesis in Drosophila melanogaster is characterized by a specific transcriptional program during the spermatocyte stage. Transcription of thousands of genes is regulated by the interaction of several proteins or complexes, including a tTAF-containing TFIID variant, tMAC, Mediator, and chromatin interactors, e.g., bromodomain proteins. We addressed how distinct subsets of target genes are selected. We characterized the highly similar proteins tPlus3a and tPlus3b, which contain a Plus3 domain and are enriched in the testis, mainly in spermatocytes. In tPlus3a and tplus3b deletion mutants generated using the CRISPR/Cas9 system, fertility was severely reduced and sperm showed defects during individualization. tPlus3a and tPlus3b heterodimerized with the bromodomain protein tBRD-1. To elucidate the role of the tPlus3a and tPlus3b proteins in transcriptional regulation, we determined the transcriptomes of tplus3a-tplus3b and tbrd-1 deletion mutants using next-generation sequencing (RNA-seq) and compared them to that of the wild-type. tPlus3a and tPlus3b positively or negatively regulated the expression of nearly 400 genes; tBRD-1 regulated 1,500 genes. Nearly 200 genes were regulated by both tPlus3a and tPlus3b and tBRD-1. tPlus3a and tPlus3b activated the Y-chromosomal genes kl-3 and kl-5 , which indicates that tPlus3a and tPlus3b proteins are required for the function of distinct classes of genes. tPlus3a and tPlus3b and tBRD-1 repress genes relevant for seminal fluid and heat shock. We hypothesize that tPlus3a and tPlus3b proteins are required to specify the general transcriptional program in spermatocytes.