In most mammals, the expression of SRY (sex-determining region on the Y chromosome) initiates the development of testes, and thus determines the sex of the individual. However, despite the pivotal role of SRY, its mechanism of action remains elusive. One important missing piece of the puzzle is the identification of genes regulated by SRY. In this study we used chromatin immunoprecipitation to identify direct SRY target genes. Anti-mouse SRY antibody precipitated a region 7.5 kb upstream of the transcriptional start site of cerebellin 4 precursor (Cbln4), which encodes a secreted protein. Cbln4 is expressed in Sertoli cells in the developing gonad, with a profile mimicking that of the testis-determining gene SRY-box containing gene 9 (Sox9). In transgenic XY mouse embryos with reduced Sox9 expression, Cbln4 expression also was reduced, whereas overexpression of Sox9 in XX mice caused an upregulation of Cbln4 expression. Finally, ectopic upregulation of SRY in vivo resulted in ectopic expression of Cbln4. Our findings suggest that both SRY and SOX9 contribute to the male-specific upregulation of Cbln4 in the developing testis, and they identified a direct in vivo target gene of SRY.