In this work we applied colloidal preparation methods to synthesize AuCu nanocrystals (NCs) in the ordered tetragonal phase with an atomic composition close to 50:50. We deposited the NCs on a support (Al2O3), studied their transformations upon different redox treatments, and evaluated their catalytic activity in the CO oxidation reaction. The combined analyses by energy dispersive X-ray spectroscopy (EDX)-scanning transmission electron microscopy (STEM), selected area electron diffraction (SAED), and in situ diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) highlighted a phase segregation between gold and copper upon the high-temperature (350 °C) oxidizing treatment. While gold remained localized in the NCs, copper was finely dispersed on the support, likely in the form of oxide clusters. AuCu alloyed NCs, this time in the form of solid solution, face-centered cubic phase, were then restored upon a reducing treatment at the same temperature, and their catalytic activity was significantly enhanced in comparison to that of the oxidized system. The composition of the NCs and consequently the CO oxidation reaction rate were also affected by the CO/O2 reacting atmosphere: regardless of the pretreatment, the same catalytic activity was approached over time on stream at temperatures as low as 100 °C. Consistently, the same situation was observed on the catalyst surface as probed by EDX-STEM, SAED, and DRIFTS. All of these transformations were found to be fully reversible.