New vascular network formation is a critical step in the wound healing process and a primary limiting factor in functional tissue regeneration. Like many tissues, neovascular networks have been shown in vitro to be highly sensitive to mechanical conditions; however, the effects of matrix deformations on neovascular network formation and remodeling in engineered tissue regeneration in vivo have not been evaluated. We quantified the effects of early and delayed functional loading on neovascular growth in a rat model of large bone defect regeneration using compliant fixation plates that were unlocked to allow transfer of ambulatory loads to the defect either at the time of implantation (early), or after 4 wk of stiff fixation (delayed). Neovascular growth and bone regeneration were quantitatively evaluated 3 wk after the onset of loading by contrast-enhanced microcomputed tomography and histology. The initial vascular response to bone injury featured robust angiogenesis and collateral vessel formation, increasing parameters such as vascular volume and connectivity while decreasing degree of anisotropy. Application of early mechanical loading significantly inhibited vascular invasion into the defect by 66% and reduced bone formation by 75% in comparison to stiff plate controls. In contrast, delaying the onset of loading by 4 wk significantly enhanced bone formation by 20% and stimulated vascular remodeling by increasing the number of large vessels and decreasing the number of small vessels. Together, these data demonstrate the mechanosensitivity of neovascular networks and highlight the capacity of biomechanical stimulation to modulate postnatal vascular growth and remodeling.