An atomic force microscope was used to measure the hardness and elasticity of fully-hydrated peritubular and intertubular human dentin. The standard silicon nitride AFM tip and silicon cantilever assembly were replaced with a diamond tip and stainless steel cantilever having significantly higher stiffness. Hardness was measured as the ratio of the applied force to the projected indentation area for indentations with depths from 10–20 nm. The sample stiffness was measured by imaging specimens in a force-modulated mode. Hardness values of 2.3 ± 0.3 GPa and 0.5 ± 0.1 GPa were measured for the peritubular and intertubular dentin, respectively. Stiffness imaging revealed that the elastic modulus of the peritubular dentin was spatially homogeneous; whereas, there was considerable spatial variation in the elasticity of the intertubular dentin. The atomic force microscope can be used to measure the mechanical properties of fully hydrated calcified tissues at the submicron level of spatial resolution, thus augmenting more traditional depth sensing probes.