The hydrogen effect on a X65 carbon steel was investigated using in-situ electrochemical nanoindentation approach. The alterations in elastic behavior, pop-in load, and hardness under hydrogen-free and hydrogen-charged conditions in both ferrite and bainite were compared and discussed. The results demonstrated a non-affected elastic behavior by hydrogen in both microconstituents. The homogeneous and heterogeneous dislocation nucleation are proposed as the dominant mechanisms for pop-in behavior in ferrite and bainite, respectively. In addition, the reduction of pop-in load by hydrogen in both microconstituents indicates a hydrogen-enhanced dislocation nucleation in both homogenous and heterogeneous manners. Moreover, a hydrogen-induced hardness increment was detected in both microconstituents, which is related to the hydrogen-enhanced lattice friction on dislocations. Also, the more prominent hardness increment in bainite was caused by its significantly more trapping sites.