BackgroundDomestic geese are seasonal breeders and have the lowest reproductive capacity among all poultry species. Magang geese is a topical short-day breeder, short photoperiod exposure stimulates its reproductive activity while long photoperiod inhibits. To explore epigenetic change that could influence reproductive activity, we performed whole genome bisulfite sequencing and transcriptome sequencing in the hypothalamus at three reproductive stages during long-light exposure in male Magang geese.ResultsA total number of 10,602 differentially methylated regions (DMRs) were identified among three comparison groups. We observed that the vast majority of DMRs were enriched in intron regions. By integrating the BS-sequencing and RNA-seq data, the correlation between methylation changes of CG DMRs and expression changes of their associated genes was significant only for genes containing CG DMRs in their intron. A total of 278 DMR-associated DEGs were obtained among the three stages. KEGG analysis revealed that the DMR-associated DEGs were mainly involved in 11 pathways. Among them, the neuroactive ligand-receptor interaction pathway was significantly enriched in both two comparisons (RA vs.RD and RD vs.RI); the Wnt signaling pathway, apelin signaling pathway, melanogenesis, calcium signaling pathway, focal adhesion, and adherens junction were significantly enriched in the RA vs. RI comparison. In addition, the expression level of two serotonin-metabolic genes was significantly altered during reproductive axis inactivation by the methylation status of their promoter region (TPH2) and intron region (SLC18A2), respectively. These results were confirmed by Bisulfite sequencing PCR (BSP), pyrosequencing, and real-time qPCR, indicating that serotonin metabolic signaling may play a key role in decreasing the reproductive activity of Magang geese induced by long-light exposure. Furthermore, we performed a metabolomics approach to investigate the concentration of neurotransmitters among the three stages, and found that 5-HIAA, the last product of the serotonin metabolic pathway, was significantly decreased in the hypothalamus during RI.ConclusionsOur study reveals that the methylation status of the serotonin metabolic pathway in the hypothalamus is associated with reproductive inactivation, and provided new insight into the effect of DNA methylation on the reproductive regulation of the hypothalamus in Magang geese.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12864-023-09342-0.