No current clinical treatments provide an ideal long-term solution for repair of long bone segment defects. Incomplete healing prevents patients from returning to preinjury activity and ultimately requires additional surgery to induce healing. Obtaining autologous graft material is costly, incurs morbidity, requires surgical time, and quality material is finite. In this pilot study, 3D printed biomimetic scaffolds were used to facilitate rapid bone bridging in critical sized defects in a sheep model. An inverse trabecular pattern based on micro-CT scans of sheep trabecular bone was printed in polybutylene terephthalate. Scaffolds were coated with micron-sized tricalcium phosphate particles to induce osteoconductivity. Mesenchymal stem cells (MSCs) were isolated from sheep inguinal and tail fat, in one group of sheep and scaffolds were infiltrated with MSCs in a bioreactor. Controls did not undergo surgery for cell extraction. Scaffolds were implanted into two experimental and two control adult sheep, and followed for either 3 or 6 months. Monthly radiographs and post explant micro-CT scanning demonstrated bone formation on the lateral, anterior, medial, and posterior-medial aspects along the entire length of the defect. Bone formation was absent on the posterior-lateral aspect where a muscle is generally attached to the bone. The 3-month time point showed 15.5% more cortical bone deposition around the scaffold circumference while the 6-month time point showed 40.9% more bone deposition within scaffold pores. Control sheep failed to unite. Serum collagen type-1C-terminus telopeptides (CTX-1) showed time-dependent levels of bone resorption, and calcein labeling demonstrated an increase in bone formation rate in treated animals compared with controls. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2018.