The triboelectrification behavior of metal friction pairs is a crucial area of research for understanding the friction and wear. In this study, the triboelectrification behavior of surface nanocrystallization of medium carbon steel was investigated. Friction and wear tests were performed using a spherical-on-disc tribometer under oil-free lubrication while measuring triboelectric current signals. The results demonstrated that microstructural differences in chemically identical materials give rise to distinct charging behavior. In particular, nanocrystalline medium carbon steel surfaces exhibited significantly lower triboelectrification properties compared to their original surfaces. The main reason for the different triboelectrification behavior of the nanocrystalline medium carbon steel surfaces was attributed to the lattice distortion of nanospheres, which was explained using the theory of work function. And the underlying mechanism was experimentally verified using a D/max-Ultima+ X-ray diffractometer to measure the lattice distortion of nanospheres in medium carbon steel. Moreover, the study found a correlation between triboelectric current, friction coefficient, and loads. The findings of this study provide valuable insights into the role of surface microstructure in the friction and wear of materials.