The macrocyclic [FeIII(L1)Cl2]+ (1, L1 = N,N′-dimethyl-2,11-diaza[3,3](2,6)pyridinophane) complex is an active catalyst for the oxidation of water to oxygen using [NH4]2[CeIV(NO3)6] (CAN), NaIO4, or Oxone as the oxidant. The mechanism of 1-catalysed water oxidation was examined by spectroscopic methods and by 18O-labelling experiments, revealing that FeIVO and/or FeVO species are likely to be involved in the reaction. The redox behaviour of 1 and these high-valent FeO species of L1 has been examined by both cyclic voltammetry and density functional theory (DFT) calculations. In aqueous solutions, the cyclic voltammograms of 1 at different pH show a pH-dependent reversible couple (E1/2 = +0.46 V vs. SCE at pH 1) and an irreversible anodic wave (Epa = +1.18 V vs. SCE at pH 1) assigned to the FeIII/FeII couple and the FeIII to FeIV oxidation, respectively. DFT calculations showed that the E value of the half reaction involving [FeV(L1)(O)(OH)]2+/[FeIV(L1)(O)(OH2)]2+ is +1.42 V vs. SCE at pH 1. Using CAN as the oxidant at pH 1, the formation of an FeIVO reaction intermediate was suggested by ESI-MS and UV-vis absorption spectroscopic measurements, and the rate of oxygen evolution was linearly dependent on the concentrations of both 1 and CAN. Using NaIO4 or Oxone as the oxidant at pH 1, the rate of oxygen evolution was linearly dependent on the concentration of 1, and a reactive FeVO species with formula [FeV(L1)(O)2]+ generated by oxidation with NaIO4 or Oxone was suggested by ESI-MS measurements. DFT calculations revealed that [FeV(L1)(O)2]+ is capable of oxidizing water to oxygen with a reaction barrier of 15.7 kcal mol−1.