Electrochemical CO reduction reaction (eCORR) is of great significance to energy and environmental engineering, while fundamental questions remain regarding its mechanisms. Herein, we formulate a fundamental understanding of the interplay between the applied potential () and kinetics of CO activation in eCORR on Cu surfaces. We find that the nature of the CO activation mechanism in eCORR varies with , and it is the sequential electron-proton transfer (SEPT) mechanism dominant at the working but switched to the concerted proton-electron transfer (CPET) mechanism at highly negative . We then identify that the barrier of the electron-transfer step in the SEPT mechanism exhibits an inverted region as decreases, which originates from the rapidly rising Pauli repulsion in the physisorption of CO with decreasing . We further demonstrate catalyst designs that effectively suppress the adverse effect of Pauli repulsion. This fundamental understanding may be general for the electrochemical reduction reactions of closed-shell molecules.