Co3O4 is a well-known low temperature CO oxidation catalyst, but it suffers from rapid deactivation. We have thus examined room temperature CO oxidation on Co3O4 catalysts by combined DSC, TGA and MS measurements, as well as by pulsed chemisorption to differentiate the contributions of CO adsorption and reaction to CO2. Catalysts pretreated in oxygen at 400 ⁰C are most active, with the initial interaction of CO and Co3O4 being strongly exothermic and with maximum amounts of CO adsorption and reaction. The initially high room temperature activity levels-off within tens of minutes, suggesting that the oxidative pretreatment creates an oxygen-rich reactive Co3O4 surface that upon reaction onset loses its active oxygen. This specific active oxygen is not reestablished by gas phase O2 during the room temperature reaction. When the reaction temperature is increased to 150 ⁰C, full conversion can be maintained for 100 hours, and even after cooling back to room temperature. Apparently, deactivating species are avoided this way, whereas exposing the active surface even briefly to pure CO leads to immediate deactivation. Computational studies using DFT helped to identify the CO adsorption sites, determine oxygen vacancy formation energies and the origin of deactivation. A new species of CO bonded to oxygen vacancies at room temperature was identified, which only reacts with an O2 moiety filling a vacancy site at higher temperature (around 100⁰C). The interaction between oxygen vacancies was found to be small, so that in the active state most lattice oxygen species are available for reaction in parallel.