It has long been known that the bone adapts according to the local mechanical environment. To date, however, a model for studying the effects of functional mechanical loading on tissue-engineered bone repair in vivo has not yet been established. We have developed a rat femoral defect model, in which ambulatory loads are transduced through the implanted tissue-engineered construct to elucidate the role of the mechanical environment in functional restoration of a large bone defect. This model uses compliant fixation plates with integrated elastomeric segments, which allow transduction of ambulatory loads. Multiaxially and uniaxially compliant plates were characterized by mechanical testing and evaluated using in vivo pilot studies. In the first study, experimental limbs were implanted with multiaxial plates, which have a low stiffness in multiple loading modes. In the second study, experimental limbs were stabilized by a uniaxial plate, which allowed only axial deformation of the defect. X-ray scans and mechanical testing revealed that the multiaxial plates were insufficient to stabilize the defect and prevent fracture under ambulatory loads as a result of low flexural and torsional stiffness. The uniaxial plates, however, maintained integrity of the defect when implanted over a 12 week period. Postmortem microCT scans revealed a 19% increase in bone volume in the axially loaded limb compared with the contralateral standard control, and postmortem mechanical testing indicated that torsional strength and stiffness were increased 25.6- and 3.9-fold, respectively, compared with the control. Finite element modeling revealed high strain gradients in the soft tissue adjacent to the newly formed bone within the implanted construct. This study introduces an in vivo model for studying the effects of physiological mechanical loading on tissue-engineered bone repair. Preliminary results using this new in vivo model with the uniaxially compliant plate showed positive effects of load-bearing on functional defect repair.