<p>Tailoring interfacial structures in CuO_x/CeO₂ catalysts via pretreatment atmosphere significantly modulates their CO oxidation performance. Catalysts were pretreated under oxidation (CuCe-Oxi), reaction (CuCe-Rex), and reduction (CuCe-Red) atmospheres. CuCe-Red exhibits superior activity, achieving 66% CO conversion at 70&#xa0;°C, markedly higher than CuCe-Rex (45%) and CuCe-Oxi (33%). Crucially, the intrinsic activity (TOF) of CuCe-Red is double that of the others, directly linked to distinct interfacial site structures induced by pretreatment. <i>In-situ</i> spectroscopy and temperature-programmed techniques reveal that reductive pretreatment generates key active sites: low-coordinated Cu⁺ sites and strongly interacting [Cu^m+-O_x-Ceⁿ⁺] interfacial species (including Cu²⁺-O_L-Ce⁴⁺ and asymmetric oxygen vacancy Cu⁺-O_v-Ce³⁺ configurations). The proportion of these specific sites correlates positively with TOF. The low-coordinated Cu⁺ sites substantially enhance CO surface coverage and improve CO activation. Systematic kinetic analysis, O₂-TPO, and CO-TPD confirm the reaction follows the Mars-van Krevelen mechanism. CO surface activation is identified as the rate-determining step (RDS). Therefore, this study demonstrates that targeted manipulation of the catalyst's interfacial microenvironment through pretreatment atmosphere controls the formation of highly active low-coordinated Cu⁺ and specific [Cu^m+-O_x-Ceⁿ⁺] species. This structural evolution directly governs CO adsorption, activation, and ultimately, catalytic performance, providing deep insights into the structure–activity relationship for CuO_x/CeO₂ oxidation catalysts.</p> Graphical Abstract <p></p>