Testicular vasculogenesis is one of the key processes regulating male gonad morphogenesis. The knowledge of the molecular cues underlining this phenomenon is one of today's most challenging issues and could represent a major contribution toward a better understanding of the onset of testicular morphogenetic disorders. R-spondin 1 has been clearly established as a candidate for mammalian ovary determination. Conversely, very little information is available on the expression and role of R-spondin 1 during testicular morphogenesis. This study aims to clarify the distribution pattern of R-spondin 1 and other partners of its machinery during the entire period of testicular morphogenesis and to indicate the role of this system in testicular development. Our whole mount immunofluorescence results clearly demonstrate that R-spondin 1 is always detectable in the testicular coelomic partition, where testicular vasculature is organized, while Dickkopf-1 is never detectable in this area. Moreover, organ culture experiments of embryonic male UGRs demonstrated that Dickkopf-1 acted as an inhibitor of testis vasculature formation. Consistent with this observation, real-time PCR analyses demonstrated that DKK1 is able to slightly but significantly decrease the expression level of the endothelial marker Pecam1. The latter experiments allowed us to observe that DKK1 administration also perturbs the expression level of the Pdgf-b chain, which is consistent with some authors' observations relating this factor with prenatal testicular patterning and angiogenesis. Interestingly, the DKK1 induced inhibition of testicular angiogenesis was rescued by the co-administration of R-spondin 1. In addition, R-spondin 1 alone was sufficient to enhance, in culture, testicular angiogenesis.