Nutrient metabolism is required to maintain energy balance in animal organisms, and fatty acids play an irreplaceable role in fat metabolism. In this study, microRNA sequencing was performed on mammary gland tissues collected from cows during early, peak, and late lactation to determine miRNA expression profiles. Differentially expressed miRNA (miR-497) was selected for functional studies of fatty acid substitution. Simulants of miR-497 impaired fat metabolism [triacylglycerol (TAG) and cholesterol], whereas knockdown of miR-497 promoted fat metabolism in bovine mammary epithelial cells (BMECs) in vitro. In addition, in vitro experiments on BMECs showed that miR-497 could down-regulate C16:1, C17:1, C18:1, and C20:1 as well as long-chain polyunsaturated fats. Thus, these data expand the discovery of a critical role for miR-497 in mediating adipocyte differentiation. Through bioinformatics analysis and further validation, we identified large tumor suppressor kinase 1 (LATS1) as a target of miR-497. siRNA-LATS1increased concentrations of fatty acids, TAG, and cholesterol in cells, indicating an active role ofLATS1in milk fat metabolism. In summary, miR-497/LATS1can regulate the biological processes associated with TAG, cholesterol, and unsaturated fatty acid synthesis in cells, providing an experimental basis for further elucidating the mechanistic regulation of lipid metabolism in BMECs.