Background: Buffalo milk represents an indispensable source of nourishment, and exosomes from buffalo milk, compared with cow-milk, would yield novel miRNAs that would inform the potential physiological importance of consumption of buffalo-milk exosomes.Results: Here, we isolated exosomes from buffalo and cow milk samples that were obtained at mid-lactation period, and the exosomal miRNA profiles were then generated using RNA-seq. As control, miRNA sequencing data of pig, human and panda milk exosomes were downloaded from GEO database. Finally, a total of 27 milk exosomal miRNA profiles that included 4 buffalo, 4 cow, 8 pig, 4 human and 7 panda were analyzed using the miRDeep2 program. For these datasets there were 558 unique miRNA candidates existed across all species, and the top 10 highly expressed miRNA were evolutionarily conserved across multiple species. Functional analysis revealed that these milk enriched miRNAs targeted 400 putative sites to modulate disease resistance, immune responsiveness and basic metabolism events. In addition, a total of 32 miRNAs in buffalo milk were significantly up-regulated compared with non-buffalo milks, while 16 were significantly down-regulated. Of interest, functional analysis showed that up-regulated miRNA were mainly related to host metabolism processes, while the predicted functions of down-regulated miRNAs were enriched in immune response.Conclusion: In this study, we explored the exosomal miRNAome differences between milks of different animals, expanding the theoretical basis for potential applications of the miRNA-containing vesicles.