Hydrogen embrittlement (HE) poses a significant challenge for the development of high-strength metallic materials. However, explanations for the observed HE phenomena are still under debate. To shed light on this issue, here we investigated the hydrogen-defect interaction by comparing the dislocation structure evolution after hydrogen adsorption and desorption in a Fe-28Mn-0.3C (wt%) twinning-induced plasticity steel with an austenitic structure using in situ electron channeling contrast imaging. The results indicate that hydrogen can strongly affect dislocation activities. In detail, hydrogen can promote the formation of stacking faults with a long dissociation distance. Besides, dislocation movements are frequently observed during hydrogen desorption. The required resolved shear stress is considered to be the residual stresses rendered by hydrogen segregation. Furthermore, the microstructural heterogeneity could lead to the discrepancy of dislocation activities even within the same materials.