Electron paramagnetic resonance was used to study the donor that is responsible for the n-type conductivity in unintentionally doped (UID) β-Ga2O3 substrates. We show that in as-grown materials, the donor requires high temperature annealing to be activated. In partly activated materials with the donor concentration in the 1016 cm−3 range or lower, the donor is found to behave as a negative-U center (often called a DX center) with the negative charge state DX− lying ∼16–20 meV below the neutral charge state d0 (or Ed), which is estimated to be ∼28–29 meV below the conduction band minimum. This corresponds to a donor activation energy of Ea∼44–49 meV. In fully activated materials with the donor spin density close to ∼1 × 1018 cm−3, donor electrons become delocalized, leading to the formation of impurity bands, which reduces the donor activation energy to Ea∼15–17 meV. The results clarify the electronic structure of the dominant donor in UID β-Ga2O3 and explain the large variation in the previously reported donor activation energy.