Abstract Intrinsic defects and defect clusters play an important role in the room-temperature transport of cuprous oxide. Neutralization of these defects by doping and/or modifying the synthesis process is essential to improve the room temperature (RT) hole mobility in cuprous oxide. Towards this, we annealed polycrystalline cuprous oxide under Cu-rich conditions leading to neutralization of the intrinsic acceptor defect. The concentration of both the acceptor defects (VCu and Vsplit Cu) that are already present, reduces by four to five orders of magnitude. This is commensurate with the amount of possible Cu incorporation under different annealing conditions, indicating the back-filling of a large fraction of the Cu vacancies. Unforeseeably, the experimental conditions lead to the creation of yet another higher order extended defect (3VCu + 2Cui) with a defect level at ≈0.5 eV above the valence band. The formation of such a defect is also indirectly suggested by the analysis of carrier concentration vs. temperature data and first principle calculations. Such singly ionized higher order defects with a possibly higher capture cross-section act as more effective traps resulting in reduced hole mobility.