The effect of post-irradiation annealing on the microstructures and mechanical properties of V-4Cr-4Ti alloys was studied. Helium-hydrogen-irradiated sequentially V-4Cr-4Ti alloys at room temperature (RT) were undergone post-irradiation annealing at 450 °C over periods of up to 30 h. These samples were carried out by high-resolution transmission electron microscopy (HRTEM) observation and nanoindentation test. With the holding time, large amounts of point defects produced during irradiation at RT accumulated into large dislocation loops and then dislocation nets which promoted the irradiation hardening. Meanwhile, bubbles appeared. As annealing time extended, these bubbles grew up and merged, and finally broke up. In the process, the size of bubbles increased and the number density decreased. Microstructural changes due to post-irradiation annealing corresponded to the change of hardening. Dislocations and bubbles are co-contributed to irradiation hardening. With the holding time up to 30 h, the recovery of hardening is not obvious. The phenomenon was discussed by dispersed barrier hardening model and Friedel-Kroupa-Hirsch relationship.