Single-atom catalysts have recently been subject to considerable attention within applied catalysis. However, complications in the preparation of well-defined single-atom model systems have hampered efforts to determine the reaction mechanisms underpinning the reported activity. By means of an atomic layer deposition method utilizing the steric hindrance of the ligands, isolated Fe1O3 motifs were grown on a single-crystal Cu2O(100) surface at densities up to 0.21 sites per surface unit cell. Ambient pressure X-ray photoelectron spectroscopy shows a strong metal-support interaction with Fe in a chemical state close to 3+. Results from scanning tunneling microscopy and density functional calculations demonstrate that isolated Fe1O3 is exclusively formed and occupies a single site per surface unit cell, coordinating to two oxygen atoms from the Cu2O lattice and another through abstraction from O2. The isolated Fe1O3 motif is active for CO oxidation at 473 K. The growth method holds promise for extension to other catalytic systems.