Cancellous bone plays a crucial structural role in the skeleton, yet little is known about the microstructure-mechanical property relationships of the tissue at the microscale. Cancellous tissue is characterized by a microstructure consisting of layers interspaced with transition zones with different proportions of collagen and mineral. In this study, the quasistatic and dynamic mechanical properties of lamellar and interlamellar tissue in human vertebrae were assessed with nanoindentation, and the collagen content and organization were characterized with second harmonic generation microscopy. Lamellar tissue was 35% stiffer, 25% harder, and had a 13% lower loss tangent relative to interlamellar tissue. The stiff, hard lamellae corresponded to areas of highly ordered, collagen-rich material, with a relatively low loss tangent, whereas the compliant, soft interlamellar regions corresponded to areas of disordered or collagen-poor material. These data suggest an important role for collagen in the tissue-level mechanical properties of bone.