Previous studies have suggested that surface hardening occurs in human tooth enamel under certain loading conditions. However, the occurrence mechanism and significance remain unclear. In this study, the surface hardening behavior of enamel under masticatory loading was studied in vitro using impact treatment and the nanoindentation/scratch technique to identify the mechanism and antiwear effect. The fundamental block of enamel is made of hydroxyapatite (HAP) nanofibers, which consist of fine nanoparticles held together by protein. These fibers respond to masticatory loading in two ways: bending deflection at low loads and fragmentation at high loads. When the contact pressure exceeds the bonding strength between the nanoparticles, the HAP fibers split into fine nanoparticles and then form a surface layer consisting of tightly packed nanoparticles. This results in surface hardening dominated by an increased hardness and elastic modulus. The maximum degree and depth of surface hardening were determined as approximately 60% and 100 nm, respectively. With the occurrence of surface hardening, the wear resistance of the enamel is enhanced, which is manifested by a reduced friction coefficient and wear volume. In summary, the surface hardening of enamel induced by masticatory loading is a result of HAP nanoparticle rearrangement as a response of the enamel hierarchical structure to high chewing loads. It is adaptive overload protection derived from the enamel hierarchical structure and plays a critical role in resisting excessive wear induced by high chewing loads.