The secondary osteon is the fundamental building block of compact cortical bone at the tissue level. Light and scanning electron microscopy have shown that the osteon consists of a laminated cylindrical composite of mineralized collagen fibril lamellae ∼5–7 μm thick. Using scanning nanoindentation and quantitative backscattered electron imaging on secondary osteons from the human femoral midshaft, we found that the indentation modulus shows a periodic variation between ∼24 GPa and ∼27 GPa within a single lamella. The average lamellar value remains nearly constant across the osteon and increases abruptly to more than 30 GPa at the interstitial bone interface. The local mineral content, determined from quantitative backscattered electron imaging at the indented locations, shows also a lamellar level modulation and is positively correlated with the indentation modulus at the same tissue position. We propose that such a mechanically and compositionally modulated structure may be an effective crack-stopping mechanism in bone.