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MicroRNAs (miRNAs) are a class of non-coding single-stranded RNA molecules of about 19-25 nucleotides in length regulating different kinds of biological processes, including lipid metabolism. In this research, we explored the effect of bta-miR-33a on lipid metabolism in bovine mammary epithelial cells (BMECs) of Chinese Holstein for the first time. For this purpose, the vectors of bta-miR-33a mimic, bta-miR-33a inhibitor, and bta-miR-33a-ShNC were constructed to overexpress and repress bta-miR-33a in BMECs. These vectorsÃ¢â‚¬â„¢ transfection effects were analyzed by examining the mRNA and protein expression levels of ELOVL6 by RT-qPCR and Western Blotting. Further, the target relationship between bta-miR-33a and target gene ELOVL6 was verified by luciferase reporter assay. Also, the intracellular triglycerides were detected using a triglyceride detection kit. The results showed that bta-miR-33a directly inhibited the expression of ELOVL6 in BMECs, decreased the mRNA expression of ELOVL5, PTPLB, CPT1A, and SC4MOL, and the mRNA expression of ALOX15 was increased, which in turn promoted the content of triglycerides. In summary, the present study suggests that bta-miR-33a regulates lipid metabolism by targeting ELOVL6, which might be a potential molecular marker of milk fat composition.