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The origin of the threshold voltage (Vth) shift that occurs in ON-state biased AlGaN/GaN high-electron mobility transistors (HEMTs) is proposed in terms of experimental researches and first-principles calculations. Experimentally, ∼20% negative shift of the Vth is found for the 0.2 μm gate length T-gate AlGaN/GaN HEMT under the ON-state stress. 1/f noise measurements are carried out to investigate the evolution of defect distribution in the GaN channel, which indicates that the Vth shift is accompanied by the diminishing of the defect level of Ev + 0.8 eV. Combined with first-principles studies based on hybrid functionals defect calculations, we find that the defect evolution of O from negatively charged VGa-ON to neutral ON-H gives rise to the decrease in acceptors in the GaN channel and thus the negative shift of Vth. Our work indicates that the strengthening of oxygen-related defects in GaN during material growth and device fabrication would improve the reliability of AlGaN/GaN HEMTs. [ABSTRACT FROM AUTHOR]