We recently identified protease nexin-1 (PN-1) or serpinE2, as a possibly underestimated player in maintaining angiogenic balance. Here, we used the well-characterized postnatal vascular development of newborn mouse retina to further investigate the role and the mechanism of action of PN-1 in physiological angiogenesis. The development of retinal vasculature was analysed by endothelial cell staining with isolectin B4. PN-1-deficient (PN-1-/-) retina displayed increased vascularization in the postnatal period, with elevated capillary thickness and density, compared to their wild-type littermate (WT). Moreover, PN-1-/-retina presented more veins/arteries than WT retina. The kinetics of retinal vasculature development , retinal VEGF expression and overall retinal structure were similar in WT and PN-1-/-mice, but we observed a hyperproliferation of vascular cells in PN-1-/-retina. Expression of PN-1 was analysed by immunoblot-ting and X-Gal staining of retinas from mice expressing beta-galactosidase under a PN-1 promoter. PN-1 was highly expressed in the first week following birth and then progressively decreased to a low level in adult retina where it localized on the retinal arteries. PCR arrays performed on mouse retinal RNA identified two angiogenesis-related factors, midkine and Smad5, that were overexpressed in PN-1-/-newborn mice and this was confirmed by RT-PCR. Both the higher vascularization and the overexpres-sion of midkine and Smad5 mRNA were also observed in gastrocnemius muscle of PN-1-/-mice, suggesting that PN-1 interferes with these pathways. Together, our results demonstrate that PN-1 strongly limits physiological angiogenesis and suggest that modulation of PN-1 expression could represent a new way to regulate angiogenesis.