Hydrogen (H) is one of the important factors which causing the failure of steel materials. In this paper microscopic defect observation, hydrogen charging experiment and defect statistics of C56E2 steel were studied, the results show that H in C56E2 steel can cause inclusion / matrix interface cavity, which could be inherited to the final material , heat treatment including quenching, tempering, could not improve those micro-defects. The average inclusion/matrix interface debonding ratio (AIDR) decreased with increased the equivalent diffusion distance of hydrogen, the correlation is strong and without any uniform rule between the descending slope and H charging time. There was not any obviously rule between AIDR and H charging time, the AIDR is increasing with the increase of the first and second peak periods and the total hydrogen content, and the correlation is weak . In the process of hydrogen charging, H diffused in the direction of H concentration decreased, the H charging time (≤216 h) applied in this paper was far from the time which required for the effect of H breaking to the steel. H in the deep inner of the sample was much lower than the max which could be reach, and transformed and broken to the steel incompletely.