PH13-8Mo stainless steel has been widely used in aerospace, petroleum and marine construction, obtaining continuous investigation attention in recent years. Based on the response of a hierarchical martensite matrix and possible reversed austenite, a systematic investigation of the evolution of the toughening mechanisms in PH13-8Mo stainless steel as a function of aging temperature was carried out. It showed there was a desirable combination of high yield strength (~1.3 GPa) and V-notched impact toughness (~220 J) after aging between 540 and 550 °C. With the increase of aging temperature, the martensite matrix was recovered in terms of the refined sub-grains and higher ratio of high-angle grain boundaries (HAGBs). It should be noted there was a reversion of martensite to form austenite films subjected to aging above 540 °C; meanwhile, the NiAl precipitates maintained a well-coherent orientation with the matrix. Based on the post mortem analysis, there were three stages of the changing main toughening mechanisms: Stage I: low-temperature aging at around 510 °C, where the HAGBs contributed to the toughness by retarding the advance of cracks; Stage II: intermediate-temperature aging at around 540 °C, where the recovered laths embedded by soft austenite facilitated the improvement of toughness by synergistically increasing the advance path and blunting the crack tips; and Stage III: without the coarsening of NiAl precipitates around 560 °C, more inter-lath reversed austenite led to the optimum toughness, relying on “soft barrier” and transformation-induced plasticity (TRIP) effects.