In this study, we investigated the correlation between the pre-strain and hydrogen embrittlement (HE) mechanisms in medium-Mn steel. Intercritically annealed Fe-7Mn-0.2C-3Al (wt.%) steel, which showed a two-phase microstructure comprising α ferrite and γR retained austenite, was used as a model alloy. As the pre-strain level increased from 0 % to 45 %, the volume fraction of γR gradually decreased owing to the strain-induced α′ martensite transformation accompanied by an increase in dislocation density. The HE resistance decreased with increasing the pre-strain level because the sample with a higher pre-strain level revealed a higher amount of dissolved hydrogen, combined with a more extensive brittle fracture region owing to the enhanced diffusion and permeation of hydrogen from the reduced γR fraction. Additionally, the H-assisted crack in the sample without pre-strain was initiated and propagated from the γR grains when the strain-induced α′ phase was formed, because most of the dissolved hydrogen was concentrated in the γR grains, and these grains were predominantly deformed compared to the other phases. However, the pre-strained sample showed more pronounced multiple H-assisted cracking at the constituent phases, such as α and α′, because it exhibited relatively well-dispersed hydrogen atoms and reduced microstrain localization at the γR grains, due to the reduced γR fraction.