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Milk can mediate maternal-neonatal signal transmission by the bioactive component extracellular vesicles (EVs), which select specific types of miRNA to encapsulate. The miRNA profiling of sheep milk EVs was characterized by sequencing and compared with that of cow milk. Nanoparticle tracking analysis revealed that the concentration of sheep milk EVs was 1.3 &#177; 0.09 &#215; 10¹² particles/mL and the diameter was peaked at 131.2 &#177; 0.84 nm. Sheep milk EVs contained various small RNAs, including tRNA, Cis-regulatory element, rRNA, snRNA, other Rfam RNA, and miRNA, which held about 36% of all the small RNAs. In total, 84 types of miRNA were annotated with <i>Ovis aries</i> by miRBase (version 22.0) in sheep milk EVs, with 75 shared types of miRNAs in all samples. The miR-26a, miR-191, let-7f, let-7b and miR-10b were highly expressed both in cow and sheep milk EVs, and 14 sheep milk EV-miRNAs in the top 20, occupying 98% of the total expression, were immune-related. Although pathway analysis showed different potential functions of cow and sheep milk EV-miRNAs, there were still some shared points: lipid metabolism (phospholipase D, glycerophospholipid and glycosylphosphatidylinositol), calcium metabolism, and nerve conduction (axon guidance and synapse). This study provides reference for the bioactive components in the milk of different species.