During lactation, mammary epithelial cells secrete fat in the form of milk fat globules that originate from intracellular lipid droplets. These droplets may form de novo from the endoplasmic reticulum or be derived from existing lipid droplets; they then either grow because enzymes of triacylglycerol synthesis relocate from the reticulum to their surface, or due to fusion and fission with other droplets. The overexpression of miR-30b-5p in the developing mouse mammary gland impairs lactation, which includes an increase in lipid droplet size. This study was performed to understand the origin of this defect affecting lipid droplets observed in transgenic mice. Electron microscopy analyses revealed a fragmented and discontinued tubular network of endoplasmic reticulum in the mammary epithelial cells of transgenic mice. The milk fatty acid composition was modified, with lower levels of medium-chain saturated fatty acids and a proportional increase in long-chain monounsaturated fatty acids in transgenic versus wild-type mice. Further, investigations of microRNA targets revealed a significant downregulation of ATLASTIN 2 (a GTPase described as playing a key role in lipid droplet formation) due to miR-30b-5p overexpression. Our results suggest that the increase in lipid droplet size observed in the mammary epithelial cells of transgenic mice might result from changes to lipid droplet formation and secretion because of direct modifications to Atl2 expression and indirect changes to endoplasmic reticulum morphology resulting from the overexpression of miR-30b-5p. (C) 2019 The Authors. Published by Elsevier Inc.