NH3 synthesis is one of the most critical industrial processes. Compared to commercial iron catalysts, Ru catalysts show high intrinsic activity in this reaction but suffer from hydrogen poisoning. By loading Ru onto supports such as electrides and hydrides, the hydrogen poisoning problem can be significantly alleviated. However, relevant studies on the structural dynamics of the Ru/electride catalysts under reaction conditions are very scarce. Taking advantage of the high sensitivity to hydrogen species, it is possible to obtain insights into the structural changes during the reaction using in situ neutron techniques. In this study, we have investigated the structural evolution of the Ru/Ca2N:e– catalyst during the ammonia synthesis reaction by in situ neutron scattering (inelastic neutron scattering, INS) technique. In situ INS experiments suggest that Ca2N:e– is likely converted to the Ca2NH phase during the reaction. Unlike the previously known structure where H and N atoms are intermixed, the formed Ca2NH exhibits a segregated structure where the H and N atoms are located in different layers separated by the Ca layer. Density functional theory calculations of the reaction energetics reveal that there are minor changes in the barriers and thermodynamics of the first N hydrogenation step between the two phases (Ca2NH phase with segregated H/N layers and intermixed Ca2NH phase), suggesting the impact of the phases on the reaction kinetics to be relatively minimal.