The prevailing view of the deactivation of carbon-supported Ru catalyst in ammonia synthesis is carbon methanation, but those strategies focusing on inhibiting carbon methanation cannot effectively solve the challenge of Ru/C catalysts. Herein we prepared four K-promoted Ru catalysts with different Ru loadings and treated the samples at 450 or 500 °C under 1 MPa during ammonia synthesis reaction. The result showed that the presence of a larger amount of Ru species enhanced methane formation at the reaction temperature, which was irrelevant to the loss in activity of ammonia synthesis. On the contrary, CO formation along with a great decline in catalytic activity can be observed when Ru catalysts are heated at 500 °C. The characterization results indicated that the severe depletion of surface oxygen groups following the CO-formation pathway, resulting in the notable decrease in the amount of gas adsorption, contributed to the deactivation of carbon-supported Ru catalyst.