MicroRNAs (miRNA) are a class of small noncoding RNAs and act as important post-transcriptional regulators of gene expression. ACSL1 is an important gene in milk lipid synthesis and STAT5a is one of the key genes in milk protein synthesis. In a previous study dealing with incubation of stearic acid in bovine mammary epithelial cells, an opposite expression pattern was observed between ACSL1 and miR-181a, STAT5a and miR-194. ACSL1 and STAT5a were also predicted as potential target genes of miR-181a and miR-194 respectively by TargetScan and PicTar, which are the most popular target gene prediction softwares. The objective of this work was to determine the potential function of miR-181a and miR-194 on milk fat and protein synthesis by defining the regulatory relationship between miR-181a and ACSL1, miR-194 and STAT5A. In the study, primary bovine mammary epithelial cells harvested from mid-lactation cows were cultured in DMEM/F12 medium added with 10% FBS and lactational hormones. Cells were transfected with miR-181a mimic, miR-181a inhibitor, miR-194 mimic and miR-194 inhibitor, and non-transfected cells were set as control. After an 48 h treatment, expression of miR-181a, miR-194, ACSL1, STAT5a were determined by real-time reverse-transcription PCR. The results showed that the overexpression of miR-181a and miR-194 inhibited the expression of ACSL1 and STAT5a respectively (P < 0.05), while the downregulation of the 2 miRNAs increased ACSL1 and STAT5a expression (P < 0.05). Therefore suggesting that miR-181a and miR-194 may play important roles in milk fat and protein synthesis.