The focus of this study is to analyze hydrogen embrittlement susceptibility of a modified AISI 4130 steel by means of incremental step loading tests. Three different microstructures with a hardness of 40 HRC were analyzed: martensite with large and small prior austenite grains and dual-phase (martensite/ferrite). According to the results, the dual-phase microstructure presented the lowest hydrogen embrittlement susceptibility and martensite with large prior austenite grains, the highest. This behavior was attributed to the lower fraction of high-angle boundaries presented by the martensite with large prior austenite grains, which led to a higher diffusible hydrogen content. Moreover, the ferrite local deformation in the dual-phase microstructure enhanced its hydrogen embrittlement resistance by lowering the stress concentration. A synergic effect of decohesion and localized plasticity was identified on the hydrogen induced fracture of the tested microstructures leading to an intergranular + quasi-cleavage fracture in the martensite and quasi-cleavage in the dual-phase microstructure. •Evaluation of the microstructure effect on hydrogen embrittlement by means of ISL test.•Hydrogen embrittlement analysis of martensitic and dual-phase microstructures.•Role of prior austenite grain size on hydrogen embrittlement susceptibility.•Effect of high-angle boundaries on hydrogen diffusion behavior.•Identification of hydrogen induced fracture micromechanisms through SEM and EBSD analysis.