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OBJECTIVES Understanding the molecular mechanisms of lipid synthesis in the mammary gland is crucial for regulating the level and composition of lipids in milk. This study aimed to investigate the functional and molecular mechanisms of miR-204-5p in mammary epithelial cells to provide a theoretical basis for milk lipid synthesis. METHODS Real-time quantitative PCR was performed to detect the transcriptional levels of miR-204-5p and related mRNA abundance in mammary epithelial cells. Western blotting was conducted to determine protein expression. Cell proliferation was assessed by Cell Counting Kit-8. A dual-luciferase reporter assay was conducted to verify the targeting relationship between miR-204-5p and SIRT1. siRNA and overexpression plasmids were transfected into mouse HC11 mammary epithelial cells. RESULTS The abundance of miR-204-5p was much higher in lactating mouse mammary glands than in other tissues, which indicated that miR-204-5p may be involved in regulating milk production. MiR-204-5p affected the expression of β-casein and milk lipid synthesis in HC11 mouse mammary epithelial cells but did not influence the proliferation of HC11 cells. Overexpression of miR-204-5p significantly increased the number of Oil Red O+ cells, triglyceride accumulation and the expression of markers associated with lipid synthesis, including FASN and PPARγ, whereas inhibition of miR-204-5p had the opposite effect. miR-204-5p promotes lipid synthesis by negatively regulating SIRT1. Overexpression of SIRT1 can repress the promotion of miR-204-5p on lipid synthesis. CONCLUSION Our findings showed that miR-204-5p can promote the synthesis of milk lipids in mammary epithelial cells by targeting SIRT1.