【Abstract】 Objective This study aimed to construct AnxA5 gene knockout mice (AnxA5-/-) using the CRISPR/Cas9 system and investigate the regulatory role of AnxA5 gene in cognition along with its underlying mechanisms. Methods AnxA5 knockout mice (AnxA5-/-) were constructed using the CRISPR/Cas9 system, and behavioral tests including water maze, Y-maze, mining test, and novel object recognition were employed to validate AnxA5's cognitive regulation. Western blotting, qPCR, immunofluorescence, and RT-CA experiments were conducted to examine AnxA5's regulatory effects on HT-22 cells and its mechanisms.Results Compared with wild-type C57 mice (WT), AnxA5 knockout mice (AnxA5-/-) exhibited reduced cognitive function. The proliferation capacity of HT-22 cells was weakened when AnxA5 was disrupted compared to the control group and siRNA-NC group. Additionally, AnxA5 deficiency activated the P65 signaling pathway. Conclusions AnxA5 deletion exerts inhibitory effects on mouse cognition through the P65 signaling pathway.