The Vth stability and gate reliability of AlGaN/GaN metal–insulator–semiconductor high-electron-mobility transistors (MIS-HEMTs) with alternating O2 plasma treatment were systematically investigated in this article. It was found that the conduction band offset at the Al2O3/AlGaN interface was elevated to 2.4 eV, which contributed to the suppressed gate leakage current. The time-dependent dielectric breakdown (TDDB) test results showed that the ALD-Al2O3 with the alternating O2 plasma treatment had better quality and reliability. The AlGaN/GaN MIS-HEMT with the alternating O2 plasma treatment demonstrated remarkable advantages in higher Vth stability under high-temperature and long-term gate bias stress.