The mutations of testicular insulin-like 3 (INSL3) hormone or its receptor RXFP2 cause cryptorchidism in male mice. Here we have examined Rxfp2 gene expression at different stages of embryonic and postnatal mouse development in male reproductive tissues employing quantitative RT-PCR and several RXFP2-specific antibodies directed toward different parts of the RXFP2 protein. Receptor expression was markedly increased after birth and was readily detectable in the epididymis, Leydig cells, and germ cells of the testis. The strongest expression was detected in adult mouse cremaster muscle. INSL3 treatment increased cell proliferation of embryonic gubernacular and TM3 embryonic Leydig cells, implicating active INSL3-mediated autocrine signaling in these cells and identifying TM3 as a novel in vitro model to study the effects of RXFP2 signaling. We generated Tg(Rxfp2-cre)Aia (Rxfp2-iCre) transgenic mice expressing improved Cre recombinase (iCre) under the control of the 2.4-kb mouse Rxfp2 promoter. The iCre was expressed in the gubernacular ligament at E14.5, indicating that this promoter is able to drive Rxfp2 gene expression during transabdominal testis descent. We demonstrated that the transcription factor Sox9, a known male sex determination factor, is expressed in mouse embryonic gubernacula and upregulated human, but not mouse, promoter luciferase reporter constructs. In conclusion, we have determined the developmental expression profile of INSL3 receptor employing newly characterized RXFP2 antisera and a novel Rxfp2-iCre transgenic mouse model. We determined the promoter region capable of providing the gubernacular-specific expression of Rxfp2. Analysis of RXFP2 promoter identified SOX9 as a new transcriptional enhancer of human gene expression.