Mammary gland (MG) is a complex secretory organ with a capability to synthesize and secrete large quantities of milk components regulated by nutrition. Negative energy balance (NEB) induced by 48-h of feed deprivation altered milk production and composition, mammary transcriptome and miRNome, in lactating goats [Ollier et al., 2007; Mobuchon et al., 2016]. However, the molecular mechanisms underlying the nutritional regulation of gene expression in MG is not fully understood. The objective of our study was to investigate the effects of a NEB induced by feed restriction on miRNAs and mRNAs of mammary gland of midlactation Holstein cows. Milk production and composition of multiparous mid-lactation (165 ± 21 DIM) Holstein cows were analyzed. Five Holstein cows underwent 6 days of feed restriction during which feed allowance was reduced to meet 50% of their net energy for lactation requirements. MG biopsies were performed before and at the end of the restriction period. Total RNAs were extracted with miRVana kit. Transcriptome analyses were performed using microarray analyses. MiRNAs were sequenced by RNAseq using Illumina HiSeq 4000. The sequences were mapped and annotated using miRDeep2 after trimming of adaptor sequences to identify known and predicted miRNAs. Statistical analyses were performed using DESeq2 package of R. Significance was considered at padj≤0.05. Bioinformatics analyses were performed using miRWalk, Enrichr, Revigo, and Venny 2.1 software. Feed restriction induced a NEB and decreased milk production, fat and protein yields, in line with previous studies [Gross et al., 2011, Pires et al 2016]. Feed restriction modified the expression of 27 miRNAs and 374 mRNAs in MG of Holstein cows. The 8 most expressed miRNAs are potentially involved in lipid metabolism, mammary remodeling and stress response. A comparison between the mRNAs targeted by the 8 most expressed miRNAs and 374 differentially expressed mRNAs identified 59 mRNAs in common. The bioinformatics analyses of these 59 mRNAs revealed their implication in lipid metabolism and endothelial cell proliferation. These effects of feed restriction on MG miRNAs and mRNAs observed in Holstein cows suggest a potential role of miRNAs in MG structure and lipid biosynthesis that could explain changes in milk production and milk component biosynthesis. Funding: This research was funded in part by the GISA program of INRA (LongHealth project) and by FEDER and Région Auvergne S3 project no. 23000794. References: Ollier et al. J Nutr. 2007;137: 560–567. doi:10.1093/jn/137.3.560 Mobuchon et al. PLOS ONE. 2015;10: e0140111. doi:10.1371/journal.pone.0140111