We developed an automatic method to characterize mice bone architecture from three-dimensional (3D) microtomographic images. The distal metaphyses of the femur of mice were imaged using 3D synchrotron radiation microtomography at the European Synchrotron Radiation Facility (ID19) with a voxel size of 6.65 μm. Within each reconstructed volume, a region of interest was defined and trabecular and cortical bones were automatically separated. Then, 3D morphologic and topologic model-independent parameters quantifying the 3D bone architecture were computed in both regions. The technique was applied to study the response of the C57BL/6J@Ico strain of mice submitted to a model of bone loss by hind limb unloading produced by tail-suspension.