In this study, we investigate the degradation characteristics of E-mode GaN High Electron Mobility Transistors (HEMTs) with a p-GaN gate by designed pulsed and prolonged negative gate (VGS) bias stress. Device transfer and transconductance, output, and gate-leakage characteristics were studied in detail, before and after each pulsed and prolonged negative VGS bias stress. We found that the gradual degradation of electrical parameters, such as threshold voltage (VTH) shift, on-state resistance (RDS-ON) increase, transconductance max (Gm, max) decrease, and gate leakage current (IGS-Leakage) increase, is caused by negative VGS bias stress time evolution and magnitude of stress voltage. The significance of electron trapping effects was revealed from the VTH shift or instability and other parameter degradation under different stress voltages. The degradation mechanism behind the DC characteristics could be assigned to the formation of hole deficiency at p-GaN region and trapping process at the p-GaN/AlGaN hetero-interface, which induces a change in the electric potential distribution at the gate region. The design and application of E-mode GaN with p-GaN gate power devices still need such a reliability investigation for significant credibility.