High-manganese twinning-induced plasticity (TWIP) steels exhibit high strain hardening, high tensile strength, and high ductility, which make them attractive for structural applications. At low tensile strain rates, TWIP steels are prone to hydrogen embrittlement (HE). Here though, we study the hardening and strengthening resulting from electrochemical hydrogen-charging of a surface layer of a Fe-26.9Mn-0.28C (wt.%) TWIP steel. We observed a 20% increase in yield strength following the electrochemical hydrogen-charging, accompanied by a reduction in ductility from 75% to 10% at a tensile strain rate of 10−3s−1. The microstructural evolution during tensile deformation was examined at strain levels of 3%, 5% and 7% by electron backscatter diffraction (EBSD) and electron channeling contrast imaging (ECCI) to study the dislocation structure of the hardened region. As expected, the microstructure of the hydrogen-hardened and the uncharged regions of the material evolve differently. The uncharged areas show entangled dislocation structures, indicating slip from a limited number of potentially coplanar slip systems. In contrast, hydrogen segregated to the grain boundaries, revealed by the deuterium-labelled atom probe tomography, delays the dislocation nucleation by blocking dislocation sources at the grain boundaries. The charged areas hence first show the formation of cells, indicating dislocation entanglement from more non-coplanar slip systems. With increasing strain, these cells dissolve, and stacking faults and strain-induced ε-martensite are formed, promoted by the presence of hydrogen. The influence of hydrogen on dislocation structures and the overall deformation mechanism is discussed in details.