〔Abstract〕 Objective To screen the differential genes and pathways of female and male C57 BL/6 J mice by transcriptome sequencing, and to lay a foundation for further exploring the molecular mechanism of behavioral differences between male and female mice. Methods 8-week-old female and male mice of the C57 BL/6 J strain were completely isolated from the mouse hippocampus, and total RNA was extracted and reverse transcribed to construct a cDNA library, and 50 single-ended mice were generated on the BGIseq500 platform(BGl-Shenzhen, China) The base reads were transcriptome sequenced. After sequencing, based on GO and KEGG databases, combined with the phyper function in R language to screen, correct and enrich the data, calculatePvalue, and then perform FDR correction onPvalue to obtainQvalue and analyze different annotated genes based on the hypergeometric test method. The expression status was analyzed, and the differential genes and pathways in the hippocampus of mice of different genders were screened out. Results Through the comparison of male and female differences, 325 differential genes were screened, including 233 up-regulated genes and 92 down-regulated genes. The functions of these differential genes were mainly enriched in long-term potentiation(LTP), calcium signal pathway, nicotine addiction and other processes. There were 362 junctions and 1 703 interaction edges in the female-male differential gene interaction network, and 10 core genes selected: lysine demethylase 5 D(Kdm5 d), cyclin-dependent kinase 5(Cdkl5), Cell output mediator(Cle1), dopamine receptor 6 a3(Slc6 a3), cassette transcription factor(Fox), precursor mRNA processing factor 4 B(Prpf4 b), glycine receptor 4(Glur4), calcium ion receptor 2(Camk2), DNA and RNA binding protein family(Son), serotonin receptor 5 b(Htr5 b). Conclusion The selected differential genes and signal pathways may lay the foundation for explaining the molecular mechanism of the differences in behavior between male and female mice.