Objective— Collateral vessel formation can functionally compensate for obstructive vascular lesions in patients with atherosclerosis. Neovascularization processes are triggered by fluid shear stress, hypoxia, growth factors, chemokines, proteases, and inflammation, as well as reactive oxygen species, in response to ischemia. Polymerase δ-interacting protein 2 (Poldip2) is a multifunctional protein that regulates focal adhesion turnover and vascular smooth muscle cell migration and modifies extracellular matrix composition. We, therefore, tested the hypothesis that loss of Poldip2 impairs collateral formation. Approach and Results— The mouse hindlimb ischemia model has been used to understand mechanisms involved in postnatal blood vessel formation. Poldip2 ^+/− mice were subjected to femoral artery excision, and functional and morphological analysis of blood vessel formation was performed after injury. Heterozygous deletion of Poldip2 decreased the blood flow recovery and spontaneous running activity at 21 days after injury. H ₂ O ₂ production, as well as the activity of matrix metalloproteinases-2 and -9, was reduced in these animals compared with Poldip2 ^+/+ mice. Infiltration of macrophages in the peri-injury muscle was also decreased; however, macrophage phenotype was similar between genotypes. In addition, the formation of capillaries and arterioles was impaired, as was angiogenesis, in agreement with a decrease in proliferation observed in endothelial cells treated with small interfering RNA against Poldip2. Finally, regression of newly formed vessels and apoptosis was more pronounced in Poldip2 ^+/− mice. Conclusions— Together, these results suggest that Poldip2 promotes ischemia-induced collateral vessel formation via multiple mechanisms that likely involve reactive oxygen species–dependent activation of matrix metalloproteinase activity, as well as enhanced vascular cell growth and survival.