Purpose: The better understanding of vertebral mechanical properties can help to improve the diagnosis of vertebral fractures. As the bone mechanical competence depends not only from bone mineral density (BMD) but also from bone quality, the goal of the present study was to investigate the anisotropic indentation moduli of the different sub-structures of the healthy human vertebral body and spondylophytes by means of microindentation. Methods: Six human vertebral bodies and five osteophytes (spondylophytes) were collected and prepared for microindentation test. In particular, indentations were performed on bone structural units of the cortical shell (along axial, circumferential and radial directions), of the endplates (along the anterio-posterior and lateral directions), of the trabecular bone (along the axial and transverse directions) and of the spondylophytes (along the axial direction). A total of 3164 indentations down to a maximum depth of 2.5. μm were performed and the indentation modulus was computed for each measurement. Results: The cortical shell showed an orthotropic behavior (indentation modulus, Ei, higher if measured along the axial direction, 14.6±2.8. GPa, compared to the circumferential one, 12.3±3.5. GPa, and radial one, 8.3±3.1. GPa). Moreover, the cortical endplates (similar Ei along the antero-posterior, 13.0±2.9. GPa, and along the lateral, 12.0±3.0. GPa, directions) and the trabecular bone (Ei= 13.7±3.4. GPa along the axial direction versus Ei=10.9±3.7. GPa along the transverse one) showed transversal isotropy behavior. Furthermore, the spondylophytes showed the lower mechanical properties measured along the axial direction (Ei=10.5±3.3. GPa). Conclusions: The original results presented in this study improve our understanding of vertebral biomechanics and can be helpful to define the material properties of the vertebral substructures in computational models such as FE analysis. © 2013.