Severe traumatic injuries often result in critical size bone defects, which are unable to heal without treatment. Autologous grafting is the standard of care but requires additional surgeries for graft procurement. Amnion-derived multipotent progenitor cells release a secretome of biomolecules identified as integral to the process of bone regeneration and angiogenesis. This secretome is currently under development as a biotherapeutic. The efficacy of this secretome biotherapeutic was evaluated in vitro on the proliferation and migration of mesenchymal stem cells and osteoprogenitor cells as well as in vivo using a critical size rat calvarial defect model. The secretome biotherapeutic was loaded onto a collagen scaffold and placed into the defect, which was allowed to heal for 4 and 12 weeks. The secretome biotherapeutic enhanced the proliferation and migration of mesenchymal stem cells and proliferation of osteoprogenitor cells. Further, the secretome biotherapeutic improved new bone volume and connectivity by 12 weeks and significantly improved angiogenesis at 4 weeks and bone density at 4 and 12 weeks with no deleterious effects. The improvement in new bone volume, connectivity, and angiogenesis suggests that the secretome biotherapeutic has beneficial effects for bone healing and a higher dose of the secretome biotherapeutic may further improve regeneration.