The luminescence of the well-known spinel-structured functional compound MgAl2O4 was studied at cryogenic temperatures below 10 K in a wide spectral range from near-IR to VUV under synchrotron radiation and electron beam excitation. The most intense luminescence band at ∼5 eV, which origin remained largely unknown up to now, showed characteristic behaviour of the donor-acceptor pair (DAP) centres and was assigned to the so-called antisite defects. The antisite DAPs are formed by position-swapped cations AlMg● and MgAl′, which define the inversion degree of the spinel structure and, to the best of our knowledge, always exceed 10% (>1.5 × 1021 cm−3) in the synthetic solids. Full data set allowed confirming that extremely high concentration of these intrinsic defects is the main reason for the strong 5-eV luminescence, which mechanism includes the electron transfer from MgAl′ to AlMg● (excitation) following by that from VO● to MgAl× (emission). Irradiation with He + ions up to fluences of 1017 cm−2 randomly displaced cations to empty sites of the spinel lattice resulting in more intragap defect levels, which induce a non-radiative energy relaxation channel quenching closely spaced while unaffecting distant DAP luminescence.