Spermatogenesis is absolutely dependent on FSH and androgens; suppression of these hormones inhibits germ cell development and thus sperm production. The final release of spermatids by the Sertoli cell, a process known as spermiation, is particularly sensitive to hormone suppression. To define the molecular mechanisms that mediate FSH and androgen effects in the Sertoli cell, we investigated the expression and regulation of micro-RNAs (miRNAs), small noncoding RNAs that regulate protein translation and modify cellular responses. By array analysis, we identified 23 miRNAs up-regulated more than 2-fold after hormone suppression in vivo and in vitro in primary Sertoli cell cultures. The regulation of four of these miRNAs (miR-23b, -30c, -30d, and -690) was confirmed by quantitative RT-PCR. Bioinformatic analysis of potential targets of hormonally regulated miRNAs identified genes important for focal adhesion and regulation of the actin cytoskeleton, processes known to be intimately associated with adhesion of spermatids to Sertoli cells. Two of the identified genes, Pten, an intracellular phosphatase, and Eps15, a mediator of endocytosis, were down-regulated by the withdrawal of hormones in vivo and possess miR-23b target sites in their 3'-untranslated regions. Overexpression of miR-23b in vitro resulted in decreased translation of PTEN and EPS15 protein as assessed by Western blot and luciferase analysis. We conclude that FSH and androgens act on Sertoli cells in stage VIII to control the expression of miRNAs that operate in a coordinated manner to regulate cell adhesion pathways and male fertility and that miRNA transcription is a new paradigm in the hormone dependence of spermiation.