The effect of H2 activation on the performance of CuFeOx catalyst for low-temperature CO oxidation was investigated. The characterizations of XRD, XPS, H2-TPR, O2-TPD, and in situ DRIFTS were employed to establish the relationship between physicochemical property and catalytic activity. The results showed that the CuFeOx catalyst activated with H2 at 100 °C displayed higher performance, which achieved 99.6% CO conversion at 175 °C. In addition, the H2 activation promoted the generation of Fe2+ species, and more oxygen vacancy could be formation with higher concentration of Oα species, which improved the migration rate of oxygen species in the reaction process. Furthermore, the reducibility of the catalyst was enhanced significantly, which increased the low-temperature activity. Moreover, the in situ DRIFTS experiments revealed that the reaction pathway of CO oxidation followed MvK mechanism at low temperature (<175 °C), and both MvK and L-H mechanism was involved at high temperature. The Cu+-CO and carbonate species were the main reactive intermediates, and the H2 activation increased the concentration of Cu+ species and accelerated the decomposition carbonate species, thus improving the catalytic performance effectively.