Vacancy defects on an iron surface have a great influence on the occurrence of hydrogen embrittlement. The adsorption/dissociation mechanism of H2S and the diffusion behavior of H atoms were calculated by first-principles spin-polarization density functional theory (DFT) on defect-free and vacancy-defective Fe(100) surfaces. The results show that the maximum dissociation energy barriers of H2S on the Fe(100) surface of defect-free and first-layer vacancy-defective Fe are 0.35 and 0.17 eV, respectively, indicating that the reactivity of the vacancy-defective Fe(100) surface is moderately increased. The existence of vacancy defects changes the preferential H atom diffusion entrance to the subsurface and shortens the diffusion path. For H diffusion in bulk Fe(100), it is found that H atoms diffuse via a tortuous path from one tetrahedral-site to a neighboring tetrahedral-site rather than diffusing through a linear trajectory. Moreover, the previously suggested path via the octahedral site is excluded due to its higher barrier and the rank of the saddle point. Diffusion barriers computed for H atom penetration from the surface into the inner-layers are approximately 0.54 eV (except for second-layer vacancy defects), which are all greater than the activation energy for dissociation of H2S on the Fe(100) surfaces. This suggests that H diffusion is more probable than H2S dissociation as the rate-limiting step for hydrogen permeation into the bulk Fe(100).