CO oxidation has been studied for more than a century; however, molecular-level understanding of its activation protocol and related intermediates remains elusive. Here, we present a unified mechanistic and kinetic picture of various electronic metal–support interactions within platinum–carbon catalysts via in situ spectroscopic/kinetic analyses and multi-scale simulations. Transient kinetic analysis and molecular dynamics simulations with a reactive force field provided a quantitative description of the competition between the oxygen association and oxygen dissociation mechanisms tuned by the interfacial charge distribution and CO coverage. Steady-state isotopic transient kinetic analysis and density functional theory calculations revealed a simultaneous shift in the rate-determining step (RDS) from O2* dissociation to O* and CO* and O2* and CO* association. A de novo strategy from the interfacial charge distribution to the reaction mechanism, kinetics/thermodynamics of RDS, and, ultimately, catalytic performance was developed to quantitatively map the above CO activation mechanism with an order-of-magnitude increase in reactivity. The proposed catalytic picture and de novo strategy are expected to prompt the development of theories and methodologies for heterogeneous catalysis.