Systemic experimental research was carried out on high-cycle fatigue behavior of FV520B steel based on the theory and calculation model of intrinsic dissipation. The experiment results show that the intrinsic dissipation of FV520B steel increases with the increase of the applied stress amplitude. Generally, the inflection point of intrinsic dissipation corresponds to the transition of the generation mechanism of intrinsic dissipation: from the reversible motion of the microstructure (the swing of dislocation lines between strong pinning points) to the combined effects of the reversible and irreversible motion of the microstructure (the generation of permanent slip, the unpinning from strong points and the multiplication of dislocation). And the stress amplitude corresponding to the inflection point is just the critical stress value inducing fatigue damage accumulation. Moreover, the results also indicate that FV520B steel subjected to constant stress amplitude keeps a relatively steady rate related to the applied stress amplitude and independent of the loading sequences. Additionally, the loading frequency has no effect on the fatigue damage per loading cycle. Fatigue failure will occur once the amount of the intrinsic dissipation, due to the irreversible motion of the microstructure, accumulates to a threshold value. And the energy threshold is found to be independent of the loading history.