Mammary epithelial cells are regulated by steroid hormones, growth factors, and even microRNAs. MiR-15b has been found to regulate lipid metabolism in adipocytes; however its effects on lipid metabolism in mammary epithelial cells, the cells of lipid synthesis and secretion, are as yet unknown. The main purpose of this investigation was to explore the effect of miR-15b on lipid metabolism in mammary epithelial cells, along with the underlying mechanisms. MiR-15b was overexpressed or inhibited by miRNA mimics or inhibitors; subsequently lipid formation in mammary epithelial cells, and proteins related to lipid metabolism, were investigated. Through overexpression or inhibition of miR-15b expression, the current investigation found that miR-15b down-regulates lipid metabolism in mammary epithelial cells and is expressed differentially at various stages of mouse and goat mammary gland development. Inhibition of miR-15b expression increased lipid content in mammary epithelial cells through elevation of the lipid synthesis enzyme FASN, and overexpression of miR-15b reduced lipid content in mammary epithelial cells with decreasing levels of FASN. Moreover, the steroid hormones estradiol and progesterone decreased miR-15b expression with a subsequent increase in lipid formation in mammary epithelial cells. The expression of miR-15b was lower during lactation and negatively correlated with lipid synthesis proteins, which suggests that it may be involved in lipid synthesis and milk production. MiR-15b might be a useful target for altering lipid production and milk yield.