Cu-based p-type semiconducting oxides have been sought for water-reduction photocathodes to enhance the energy-conversion efficiency in photoelectrochemical cells. CuBi2O4 has recently attracted notable attention as a new family of p-type oxides, based on its adequate band gap. Although the identification of a major defect structure should be the first step toward understanding the electronic conduction behavior, no direct experimental analysis has been carried out yet. Using atomic-scale scanning transmission electron microscopy together with chemical probing, we identify a substantial amount of Bi-Cu-Cu-Bi antisite intermixing as a major point-defect type. Our density functional theory calculations also show that antisite Bi-Cu can seriously hinder the hole-polaron hopping between Cu, in agreement with lower conductivity and a larger thermal activation barrier under a higher degree of intermixing. These findings highlight the value of the direct identification of point defects for a better understanding of electronic properties in complex oxides.