BACKGROUND: Photoperiod is one of the important factors affecting seasonal estrus of sheep. The importance of the uterus to reproduction is self-evident. However, the uterine molecular mechanisms involved in photoperiodic regulation of seasonal estrus events in sheep remain poorly understood. In recent years, the role of uterine microRNA (miRNA) in mammalian reproduction has been continuously revealed, and it is necessary to analyze and discuss their participation in the seasonal estrus of sheep. RESULTS: We compared the miRNA expression profile in uterine tissues of Sunit sheep at 3 different photoperiods, short photoperiod (SP), short transfer to long photoperiod (SLP), and long photoperiod (LP). The results showed that 31, 29 and 21 differentially expressed miRNA (DEM) were identified between SP and LP, SP and SLP, LP and SLP, respectively. Subsequently, we constructed co-expression networks of DEM and target genes in different periods. Among all DEM, nove_320, nove_338 and nove_339 target the largest number of mRNAs. Functional annotation analysis showed that these DEM target genes can be enriched in multiple GO and KEGG signaling pathways, such as cell proliferation, apoptosis, reproductive process and biological adhesion, which are related to animal reproduction and uterine receptivity. Finally, the expression level of DEM was verified by RT-qPCR, and the binding regulation relationship between DEM and their target genes in the co-expression network was confirmed by double luciferase reporter gene experiment. CONCLUSIONS: This study revealed the changes of miRNA expression in sheep uterus under different photoperiod, providing valuable resources for understanding the molecular mechanism of sheep seasonal estrus.