Cardiovascular diseases (CVDs) are seriously threatening to human life and health. Polyunsaturated fatty acids (PUFAs) are known for their role in preventing CVDs. It is beneficial to population health to promote the content of PUFAs in bovine milk. In recent years, limited research based on molecular mechanisms has focused on this field. The biological roles of numerous microRNAs (miRNAs) remain unknown. In this study, a promising and negatively correlated pair of the miRNA (miRNA-193a-5p) and a fatty acid desaturase 1 () gene are identified and screened to explore whether they are potential factors of PUFAs' synthesis in bovine milk. The targeted relationship between miRNA-193a-5p and in bovine mammary epithelial cells (BMECs) is demonstrated by dual luciferase reporter assays. qRT-PCR and western blot assays indicate that both the expression of mRNA and the protein show a negative correlation with miRNA-193a-5p expression in BMECs. Also, miR-193a-5p expression is positively correlated with the expression of genes associated with milk fatty acid metabolism, including ELOVL fatty acid elongase 6 () and diacylglycerol O-acyltransferase 2 (). The expression of fatty acid desaturase 2 () is negatively correlated with miR-193a-5p expression in BMECs. The contents of triglycerides (TAG), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA) have a significant positive correlation with the expression of and a significant negative correlation with the expression of miR-193a-5p in BMECs. For the first time, this study confirms that miRNA-193a-5p regulates PUFAs metabolism in BMECs by targeting , indicating that miRNA-193a-5p and are underlying factors that improve PUFAs content in bovine milk.