【Abstract】Objective To construct a forebrain excitatory neuron-specific NR4A1 knockout mouse model based on the loxP-Cre recombinase system and to investigate the effects of forebrain excitatory neuron-specific NR4A1 knockout on cognitive function and anxiety-like behaviors in mice using this model. Methods Mice with the genotype NR4A1flox/+ were self-crossed, screened, and identified to obtain mice with the genotype NR4A1flox/flox, which were then crossed with Camk2a-Cre mice. Through genotyping, F1 offspring mice with the genotype NR4A1flox/+-Camk2a-Cre were obtained. Mice with this genotype were further self-crossed, and via identification using genomic DNA extracted from mice tails, mice with the genotype NR4A1flox/flox-Camk2a-Cre were screened out, which were defined as NR4A1 conditional knockout mice specifically in forebrain excitatory neurons. Cognitive ability in mice was assessed using behavioral tests including the novel object recognition test and Y-maze test, while anxiety-like behaviors were evaluated via behavioral tests such as the elevated plus maze test, open field test, and light-dark box test. Additionally, Western Blot (WB) and immunofluorescence techniques were used to detect the expression of NR4A1 in the hippocampus, a key brain region in the forebrain. Results (1) Genotyping results: Forebrain excitatory neuron-specific NR4A1 knockout mice with the genotype NR4A1flox/flox-Camk2a-Cre were successfully obtained. (2) Results of WB and immunofluorescence: WB results showed that the NR4A1 protein level in the hippocampus of knockout mice was significantly lower than that in control mice; immunofluorescence results also revealed that the intensity of NR4A1 immunofluorescence signals in the hippocampus of knockout mice was lower than that in control mice.(3) Results of cognitive behavioral tests: Compared with control mice, knockout mice exhibited no significant differences in the novel object recognition index, nor in the number of entries, distance traveled, or residence time in the novel arm of the Y-maze.(4) Results of anxiety-like behavior tests: Knockout mice spent more time in the central area of the open field than control mice, but there were no significant differences in the distance traveled or number of entries into the central area; in the light-dark box test, knockout mice had a longer residence time in the light compartment than control mice, while no significant differences were observed in the distance traveled or number of entries into the light compartment between the two groups; additionally, no statistically significant difference was found between knockout and control mice in the elevated plus maze test. Conclusion This study successfully constructed a forebrain excitatory neuron-specific NR4A1 knockout mouse model. The knockout mice showed no significant changes in cognitive level but a significant reduction in anxiety level. This model provides an important experimental tool for in-depth investigation of the role of the NR4A1 gene in physiological functions and pathological mechanisms of forebrain regions.