In this study, an electrochemical method was used to permeate hydrogen through annealed DP590 steel under various pre-strain conditions (0-15%). Stress-strain and internal friction-temperature curves of the dual phase (DP) steel were obtained from slow strain-rate tensile tests and internal friction measurements, respectively. The diffusion of interstitial atoms, formation of Cottrell atmospheres, and embrittlement mechanism of DP steel were investigated under different prestress conditions before and after hydrogen permeation. The results show that the tensile strength of DP steel first decreases and then increases and the elongation sharply decreases with increasing pre-strain. The strength and ductility present similar trends with changes in pre-strain before and after hydrogen charging, however, after hydrogen charging, an obvious increase in tensile strength and decrease in elongation are observed. Furthermore, the γ peak amplitude decreases and the Snoek-Ke-Koster (SKK) peak amplitude increases with increasing internal pre-strain according to the friction-temperature curve. The γ peak and SKK peak exhibit the same trends with increasing pre-strain before and after hydrogen charging and both the γ peak and SKK peak decrease with hydrogen charging. The dislocation density in DP steel increases after hydrogen charging.