Probing and understanding the local chemical environment of an active site is essential for designing high-performance single-atom catalysts (SACs). Density functional theory (DFT) calculations were performed to investigate the ligand configuration and site geometry of MgAl2O4-supported iridium single atoms (Ir1) toward catalytic carbon monoxide (CO) oxidation. We employed MgAl2O4(111) and MgAl2O4(211) as the model substrates with adsorbed Ir single atoms of different site geometries. DFT calculations revealed that the Mg-site on MgAl2O4(111) and the step site on MgAl2O4(211) are the most stable adsorption sites for Ir single atoms. Irrespective of site choices, CO oxidation on supported Ir single atoms follows the Eley–Rideal (E–R) mechanism, in which the surface oxygen vacancies close to the Ir single atoms activate molecular O2 with a negligible barrier and the rate-limiting step is the gas-phase CO directly attacking the O–Ir species that is modulated by a CO ligand. First-principles X-ray absorption near-edge spectra of reaction intermediates along with in situ/operando X-ray absorption spectroscopy (XAS) suggest that Ir single atoms adsorb primarily on the step sites of MgAl2O4. However, microkinetic modeling predicts that a higher activity can be attained on the equally stable Mg-site, maximizing the population of which in catalyst synthesis might prove fruitful in future studies. Electronic structure analysis indicates that the CO ligand increases the reactivity of adsorbed oxygen atoms bound to Ir single atoms by increasing the antibonding nature of the O–Ir bond.