Peripheral arterial disease (PAD) is a debilitating disease that causes insufficient blood flow to the lower extremities. It affects mainly the older population and its incidence is highly correlated with an increased risk of mortality from cardiovascular events. Upregulation of growth factors such as Vascular Endothelial Growth Factor (VEGF) is a common response to chronic tissue ischemia but this does not always result in an effective increase of blood flow. Arteriolargenesis, the remodelling of capillaries into smooth muscle containing arterioles that can regulate blood flow, is essential for function to be restored to the tissue. In this study, our aim was to investigate the role of Dll4/Notch signaling in the vessel remodelling process. We used a murine model of PAD where 8 week old C57Bl6 male mice (under 2% isoflurane anaesthesia) were subjected to femoral artery electrocoagulation on one hindlimb whilst the contralateral limb remained untouched. During surgery, the adductor muscle was injected with a combination of adenoviruses overexpressing growth factors or enhanced Green Fluorescent Protein (Ad.eGFP) for control. Blood flux to the hindpaws was measured using laser speckle contrast imaging at several time points up until day 21 after induction of ischaemia (under 2% isoflurane anaesthesia). A subset of animals was culled at day 3 after ischaemia and the ischaemic adductors were assayed using digital droplet PCR. The level of VEGF-A transcript was significantly reduced in animals injected with a combination of endothelial nitric oxide synthase and angiopoietin 1 (Ad.eNOS+Ad.Ang1) when compared with animals injected with Ad.eGFP. When animals were allowed to recover for 21 days after ischaemia, the ones injected with Ad.sDll4 had no change in blood flux compared with animals injected with Ad.eGFP. The animals injected with Ad.eNOS+Ad.Ang1 showed an enhanced blood flow recovery at earlier time points when compared to Ad.eGFP animals (day 3, 0.432±0.043 for Ad.eNOS+Ad.Ang1 vs 0.241±0.022 for Ad.eGFP), and this improvement was abrogated at later days in mice where Ad.sDll4 was also injected (day 14, 0.735±0.042 for Ad.eNOS+Ad.Ang1 vs 0.566±0.040 for Ad.eNOS+Ad.Ang1+Ad.sDll4, p<0.05 two-way repeated measures ANOVA). At the same time, mice injected with Ad.eNOS+Ad.Ang1+Ad.VEGF presented reduced levels of endogenous VEGF-A when compared to control mice, but also showed a very significant increase in Notch1 transcript relative to control and Ad.eNOS+Ad.Ang1 injected mice. Recovery from ischemia in these mice was no different from Ad.eGFP injected mice and concomitant injection of Ad.sDll4 further reduced recovery of blood flow. These results indicate that the promotion of Notch1 signaling early in the process of recovery from ischaemia is deleterious to effective, functional, vessel remodeling.