Despite recent intense interest in the development of catalysts for the electrochemical nitrogen reduction reaction (ENRR), mechanistic understanding and catalyst design principles remain lacking. In this work, we develop a strategy to determine the density of initial and steady-state active sites on ENRR catalysts that follow the Mars-van Krevelen mechanism via quantitative isotope-exchange experiments. This method allows the comparison of intrinsic activities of active sites and facilitates the identification and improvement of active-site structures for ENRR. Combined with detailed density functional theory calculations, we show that the rate-limiting step in the ENRR is likely the initial N≡N bond activation via the addition of a proton and an electron to the adsorbed N2 on the N vacancies to form N2 H. The methodology developed and mechanistic insights gained in this work could guide the rational catalyst design in the ENRR.