Objective To investigate the mechanical properties of human enamel based on resonant ultrasound spectroscopy (RUS). Methods The rectangular parallelepiped specimens of human enamel were processed. The theoretical resonant frequencies of specimens were estimated and paired with the experimental resonant frequencies measured from RUS experiments. An iterative procedure was used to adjust elastic constants of enamel until the theoretical frequencies corresponded to the experimental frequencies based on minimum mean-squared error criterion. In addition, elastic modulus, shear modulus and Poisson’s ratio were calculated respectively. Results The elastic modulus, shear modulus and Poisson’s ratio ranged from 61.52 to 80.46 GPa, 21.51 to 51.86 GPa and 0.18 to 0.43, respectively. Eliminating the effect of large specimen variances, the average of elastic modulus, shear modulus and Poisson’s ratio was 72.84 GPa, 31.94 GPa and 0.27, respectively. Conclusions RUS performs a feasibility of measuring the mechanical properties of human enamel with repeatable and nondestructive advantages. All the elastic constants and mechanical parameters can be estimated through a signal experiment. The results provide references for the development of biomimetic dental restoration materials.