The catalytic synthesis of NH 3 from the thermodynamically challenging N 2 reduction reaction under mild conditions is currently a significant problem for scientists. Accordingly, herein, we report the development of a nitrogenase-inspired inorganic-based chalcogenide system for the efficient electrochemical conversion of N 2 to NH 3 , which is comprised of the basic structure of Fe–S 2 –Mo. This material showed high activity of 8.7 mg NH 3 mg Fe −1 h −1 (24 μg NH 3 cm −2 h −1 ) with an excellent faradaic efficiency of 27% for the conversion of N 2 to NH 3 in aqueous medium. It was demonstrated that the Fe 1 single atom on Fe–S 2 –Mo under the optimal negative potential favors the reduction of N 2 to NH 3 over the competitive proton reduction to H 2 . Operando X-ray absorption and simulations combined with theoretical DFT calculations provided the first and important insights on the particular electron-mediating and catalytic roles of the Fe–S 2 –Mo motifs and Fe 1 , respectively, on this two-dimensional (2D) molecular layer slab.