In this article, electron trapping in aluminum oxide (Al2O3) thin films grown by plasma enhanced atomic layer deposition on AlGaN/GaN heterostructures has been studied and a correlation with the presence of oxygen defects in the film has been provided. Capacitance–voltage measurements revealed the occurrence of a negative charge trapping effect upon bias stress, able to fill an amount of charge traps in the bulk Al2O3 in the order of 5 × 1012 cm−2. A structural analysis based on electron energy-loss spectroscopy demonstrated the presence of low-coordinated Al cations in the Al2O3 film, which is an indication of oxygen vacancies, and can explain the electrical behavior of the film. These charge trapping effects were used for achieving thermally stable (up to 100 °C) enhancement mode operation in AlGaN/GaN transistors, by controlling the two-dimensional electron gas depletion.