The effect of hydration on the mechanical properties of osteonal bone, in directions parallel and perpendicular to the bone axis, was studied on three length scales:​ (i) the mineralized fibril level (~100 nm), (ii) the lamellar level (~6 µm);​ and (iii) the osteon level (up to ~30 µm). We used a number of techniques, namely atomic force microscopy (AFM), nanoindentation and microindentation. The mechanical properties (stiffness, modulus and/or hardness) have been studied under dry and wet conditions. On all three length scales the mechanical properties under dry conditions were found to be higher by 30–50% compared to wet conditions. Also the mechanical anisotropy, represented by the ratio between the properties in directions parallel and perpendicular to the osteon axis (anisotropy ratio, designated here by AnR), surprisingly decreased somewhat upon hydration. AFM imaging of osteonal lamellae revealed a disappearance of the distinctive lamellar structure under wet conditions. Altogether, these results suggest that a change in mineralized fibril orientation takes place upon hydration.