Biophysical stimuli are important to the development and maintenance of cancellous bone, but the regulatory mechanisms need to be understood. We investigated the effects of mechanical loading applied in vivo to native cancellous bone in the rabbit on bone formation and trabecular realignment. A novel device was developed to apply controlled compressive loads to cancellous bone in situ. The effect of loading on cancellous bone volume fraction and architecture was quantified. A 4-week experiment was performed in rabbits with devices implanted bilaterally. Cyclic 1 MPa pressures were applied daily to the right limb for 10, 25, or 50 cycles at 0.5 Hz, and the left limb served as the control without any applied loading. Microcomputed tomography and histomorphometry were used to characterize the cancellous tissue within a 4-mm spherical volume located below the loading core. In vivo cyclic loading significantly increased the bone volume fraction, direct trabecular thickness, mean intercept length, and mineral apposition rate in the loaded limbs compared with contralateral limbs. Insufficient evidence was found to demonstrate an effect of number of cycles on the cancellous adaptation between loaded and control limbs. Using a rabbit model, we demonstrated that mechanical loading applied to cancellous bone in situ increased bone formation and altered trabecular morphology. This in vivo model will allow further investigation of cancellous functional adaptation to controlled mechanical stimuli and the influence of mechanical loading parameters, metabolic status, and therapeutic agents.