The effect of hydrogen on the surface morphology and nanomechanical properties of Ni-based Alloy 725 under solution-annealed (SA) and precipitation-hardened (API) conditions was thoroughly studied. The investigation involved in situ nanoindentation testing, microscopy characterization, statistical analysis, and numerical simulation approaches. The results showed the distinctive effects of hydrogen on the pop-in and hardness in the SA and API samples. For the SA sample, hydrogen mainly dissolved as solid solute in the matrix, causing enhanced lattice friction on the dislocation motion and increasing the internal stress via lattice expansion. Thus, an enhanced hardness, a reduced pop-in width/load ratio, and numerous surface steps were detected in the presence of hydrogen. For the API sample, the strengthening γ′′ phases were the stress concentrators, and the dislocations nucleated heterogeneously, demonstrating indistinctive pop-in phenomena. Furthermore, the precipitates in the API sample affected the trapping behavior of hydrogen, thereby resulting in the hardness change, which reflected the competition between solution hardening in the matrix and vacancy softening mechanism in precipitates.