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<p>MicroRNAs (miRNAs) are non-coding single-stranded RNA molecules of about 19–25 nucleotides in length that regulate different biological processes, including lipid metabolism. In this study, 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 plasmids of bta-miR-33a mimic, bta-miR-33a inhibitor and bta-miR-33a negative control were constructed to overexpress or repress bta-miR-33a in BMECs. The effects of plasmid transfection were analysed by examining the mRNA and protein expression levels of ELOVL6 and the intracellular triglycerides. The results showed that bta-miR-33a directly inhibited the expression of <i>ELOVL6</i> in BMECs; decreased the mRNA levels of <i>ELOVL5</i>, <i>HACD2</i>, <i>CPT1A</i> and <i>MSMO1</i>; and increased the mRNA level of <i>ALOX15</i>. Sequence bta-miR-33a also increased the contents of triglycerides in the cells, presumably as a consequence of these gene expression changes. In summary, the results of the present study suggest that bta-miR-33a regulates lipid metabolism by targeting <i>ELOVL6</i>, which might be a potential molecular marker of milk fat composition.</p>