Developing transition metal-nitrogen-carbon materials (M-N-C) as electrocatalysts for the oxygen evolution reaction (OER) is significant for low-cost energy conversion systems. Further d-orbital adjustment of M center in M-N-C is beneficial to the improvement of OER performance. Herein, we synthesize a single-Mn-atom catalyst based on carbon skeleton (Mn1-N2S2Cx) with isolated Mn-N2S2 sites, which exhibits high alkaline OER activity (η10 = 280 mV), low Tafel slope (44 mV·dec−1), and excellent stability. Theoretical calculations reveal the pivotal function of isolated Mn-N2S2 sites in promoting OER, including the adsorption kinetics of intermediates and activation mechanism of active sites. The doping of S causes the increase in both charge density and work function of active Mn center, and ortho-Mn1-N2S2Cx expresses the fastest OER kinetics due to the asymmetric plane.