In this paper, the lunar soil simulant parameters were calibrated using the discrete element method (DEM) for accurate simulation of the interactions between rover wheel and lunar soil simulant. Plackett-Burman, steepest ascent, and Box-Behnken experiments were utilized to propose a parameter calibration methodology. Firstly, the Plackett-Burman experiment was adopted to verify the influences of the nine parameters on the angle of repose (AoR), while three parameters with the most significant effects were identified according to their contributions, and the steepest ascent experiment narrowed their range. Then, the Box-Behnken experiment was utilized for their calibration and obtaining their optimal values. In the simulation, the Hertz–Mindlin (with JKR) contact model was chosen as the contact model. Finally, the calibrated DEM parameters were employed to perform the AoR validation test. The relative error between the simulation and experimental results was 3.4%. The penetration tests were then accomplished further to verify the accuracy and validity of DEM parameters. The penetration resistance curves in the simulation were compatible with the experiment. The obtained results can be utilized for future studies on the interaction between the rover wheel and soil.