MicroRNAs (miRNAs) are small non-coding RNAs that are involved in mammary gland development and lactation in livestock. Little is known about the roles of miRNAs in ovine mammary gland development, hence in this study the expression profiles of miRNAs of the mammary gland tissues of ewes at peak-lactation and during the non-lactating period were investigated using RNA sequencing. A total of 147 mature miRNAs were expressed in the two periods. Compared with peak-lactation, eight miRNAs in the non-lactating ewe mammary gland were significantly up-regulated, whereas fifteen miRNAs were down-regulated. A KEGG analysis revealed that the target genes of the up-regulated miRNAs were significantly enriched in lysosome, Wnt and MAPK signaling pathways, while the target genes of down-regulated miRNAs were significantly enriched in the PI3K-Akt signaling pathway, protein processing in endoplasmic reticulum and axon guidance. These results suggest that further study of the differentially expressed miRNAs could provide a better understanding of the molecular mechanisms of mammary development and lactation in sheep. • A total of 147 mature miRNAs were expressed in the non-lactation and peak-lactation periods. • Eight up-regulated and fifteen down-regulated miRNAs were found in the non-lactation, compared to the peak-lactation. • Some KEGG pathways identified in the study are related to mammary gland development and lactation. • The RT-qPCR results from sixteen differentially expressed miRNAs confirmed the reliability and repeatability of the miRNA sequencing results. [ABSTRACT FROM AUTHOR]