A new family of ruthenium complexes based on the N-pentadentate ligand Py2Metacn (N-methyl-N′,N′′-bis(2-picolyl)-1,4,7-triazacyclononane) has been synthesised and its catalytic activity has been studied in the water-oxidation (WO) reaction. We have used chemical oxidants (ceric ammonium nitrate and NaIO4) to generate the WO intermediates [RuII(OH2)(Py2Metacn)]2+, [RuIII(OH2)(Py2Metacn)]3+, [RuIII(OH)(Py2Metacn)]2+ and [RuIV(O)(Py2Metacn)]2+, which have been characterised spectroscopically. Their relative redox and pH stability in water has been studied by using UV/Vis and NMR spectroscopies, HRMS and spectroelectrochemistry. [RuIV(O)(Py2Metacn)]2+ has a long half-life (>48 h) in water. The catalytic cycle of WO has been elucidated by using kinetic, spectroscopic, 18O-labelling and theoretical studies, and the conclusion is that the rate-determining step is a single-site water nucleophilic attack on a metal-oxo species. Moreover, [RuIV(O)(Py2Metacn)]2+ is proposed to be the resting state under catalytic conditions. By monitoring CeIV consumption, we found that the O2 evolution rate is redox-controlled and independent of the initial concentration of CeIV. Based on these facts, we propose herein that [RuIV(O)(Py2Metacn)]2+ is oxidised to [RuV(O)(Py2Metacn)]2+ prior to attack by a water molecule to give [RuIII(OOH)(Py2Metacn)]2+. Finally, it is shown that the difference in WO reactivity between the homologous iron and ruthenium [M(OH2)(Py2Metacn)]2+ (M=Ru, Fe) complexes is due to the difference in the redox stability of the key MV(O) intermediate. These results contribute to a better understanding of the WO mechanism and the differences between iron and ruthenium complexes in WO reactions ; We thank the ICIQ Foundation, MEC for a FPU PhD grant FPU14/02550 (C.C.), the European Research Foundation for projects FP7-PEOPLE-2010-ERG-268445 (J.L.F.) and ERC-2009- StG-239910 (M.C.), the MICINN for project CTQ2009-08464 (M.C.) and for a Ramon y Cajal contract, the Generalitat de Catalunya for an ICREA Academia Award (M.C.) and the CELLEX Foundation through the CELLEX-ICIQ high-throughput experimentation platform for financial support. We also thank MINECO for support through Severo Ochoa Excellence Accreditation 2014–2018 (SEV-2013-0319). We acknowledge Catexcel for a generous gift of Ts3tacn