To observe the effect of homocysteine (HCY) on the gene expression profile in rat hippocampus. Methods: Ten male SD rats were randomly divided into the control group and the high HCY group. For 14 consecutive days, high HCY group rats were injected subcutaneously with L-HCY at 60 μg/g twice a day, and control group rats were injected with equal doses of saline. The plasma HCY of each group was analyzed after establishment of model; hippocampus RNA was extracted, and sequencing was completed via BGISEQ-500 sequencing. Principal component analysis was used to compare the samples and exclude the outliers. The differentially expressed genes (DEGs) between groups were analyzed by DEGseq. According to the annotation results of KEGG and official categories, biological pathway classification and enrichment analysis were conducted on the DEGs. Results: The plasma HCY level of the high HCY group was significantly higher than that of the control group (P＜0.01). A total of 34 mRNA with significant difference in expression were screened from rat hippocampus RNA sequencing data using DEGseq. The major pathway found by biological pathway classification and enrichment analysis was the neuroactive ligand-receptor interaction pathway. Conclusion: Hyperhomocysteinemia leads to changes in the gene expression profile of rat hippocampus and interferes with the neuroactive ligand-receptor interaction and other biological pathways.