An understanding of the molecular mechanisms by which androgens drive spermatogenesis has been thwarted by the fact that few consistent androgen receptor (AR) target genes have been identified. Here, we addressed this issue using next-generation sequencing coupled with the RiboTag approach, which purifies translated mRNAs expressed in cells that express cyclic recombinase (CRE). Using RiboTag mice expressing CRE in Sertoli cells (SCs), we identified genes expressed specifically in SCs in both prepubertal and adult mice. Unexpectedly, this analysis revealed that the SC-specific gene program is already largely defined at the initiation of spermatogenesis despite the subsequent dramatic maturational changes known to occur in SCs. To identify AR-regulated genes, we generated triple-mutant mice in which the SCs express the RiboTag but lack ARs. RNA sequencing analysis revealed hundreds of SC-expressed AR-regulated genes that had previously gone unnoticed, including suppressed genes involved in ovarian development. Comparison of the SC-enriched dataset with that from the whole testes allowed us to classify genes in terms of their degree of expression in SCs. This revealed that a greater fraction of AR-up-regulated genes than AR-down-regulated genes were expressed predominantly in SCs. Our results also revealed that AR signaling in SCs causes a large number of genes not detectably expressed in SCs to undergo altered expression, thereby providing genome-wide evidence for wide-scale communication between SCs and other cells. Taken together, our results identified novel classes of genes expressed in a hormone-dependent manner in different testicular cell subsets and highlight a new approach to analyze cell type-specific gene regulation.