Simple SummaryAccurate timing of photoperiod changes is vital for animal reproduction. Previous research has shown that the polymorphisms and expression of melatonin receptor 1A (MTNR1A) play essential roles in changes in the ovine estrus cycle and seasonal reproduction. In this study, we measured the expression of MTNR1A in the ovine hypothalamus after different photoperiod treatments (short photoperiod treatment for 42 days, SP42; long photoperiod treatment for 42 days, LP42; 42 days of short photoperiod followed by 42 days of long photoperiod treatments, SP-LP42). Subsequently, the core promoter and its methylation levels at CpG sites in theMTNR1Agene in different photoperiod treatment groups were identified. We found that the core promoter region inMTNR1Awas located in a 540 bp region upstream of the transcription start site (TSS) and that the difference in the DNA methylation levels at CpG sites or the expression of MTNR1A was significantly varied in different photoperiod groups. These results suggested that the photoperiod induced the DNA methylation of theMTNR1Agene, changing its expression. The finding of photoperiod-induced DNA methylation in theMTNR1Agene is valuable for the further study of seasonal reproduction in sheep.AbstractResearch has shown that MTNR1A plays an essential role in the estrus cycle and seasonal reproduction changes in sheep. However, few people have focused on the DNA methylation of MTNR1A by season or photoperiod. In this study, using qPCR and Western blotting, we measured the MTNR1A expression in the hypothalamus of ovariectomized and estradiol-treated (OVX + E2) sheep under different photoperiod treatment conditions. Subsequently, the core promoter of theMTNR1Agene was identified, and its methylation level in sheep exposed to different photoperiod treatments was measured by pyrosequencing. The results showed thatMTNR1Agene expression significantly differed between the short 42-day photoperiod (SP42) and the LP42 or combined SP-LP42 treatment groups (p< 0.05). In addition, we determined that the coreMTNR1Apromoter region was within 540 bp upstream of the transcriptional start site (TSS) and that the DNA methylation levels at CpG sites in the SP42 vs. LP42 and SP42 vs. SP-LP42 groups significantly differed. Finally, a significant negative correlation (p< 0.001) between gene expression and DNA methylation levels was revealed, suggesting that photoperiod may induce DNA methylation of theMTNR1Agene and thus change its expression. The findings provide valuable bases for the further study of seasonal reproduction in sheep.