AbstractMilk fat depression (MFD) is characterized by a reduction in the content of milk fat, presumably caused by the anti-lipogenic effects of rumen biohydrogenation intermediates, such as trans-10 cis-12 conjugated linoleic acid (CLA). In this study, RNA-Seq technology was used to help elucidate the mammary responses involved in CLA-induced MFD in lactating ewes. To this end, we compared the milk somatic cell transcriptome of ewes suffering from CLA-induced MFD with control ewes (i.e., those without MFD), as well as with ewes fed a diet supplemented with fish oil (FO-MFD) that we previously reported affects the mammary transcriptome. In the differential expression analysis between CLA-MFD and controls, we identified 1,524 differentially expressed genes (DEGs), whereas 653 were detected between CLA- and FO-MFD groups. Although this article focuses on lipid metabolism, CLA affected the expression of many genes related to other biological processes, especially immunity. Among the 55 genes shared by both MFD conditions, some genes linked to fatty acid synthesis, such as ACACA, AACS, ACSS2, or ACSS3, were downregulated. In addition, this study provides a list of candidate genes that are not usually considered in the nutrigenomics of MFD but that may act as key regulators of this syndrome in dairy ewes.