Feeding linseed to dairy cows results in milk fat depression (MFD), but there is a wide range of sensitivity among cows. The objectives of this study were (1) To compare the mRNA expression of transcripts expressed in milk somatic cells in cows resistant or sensitive to MFD; (2) to identify metabolic pathways and transcription factors affected by MFD in resistant cows under different dietary conditions (no fat supplemented or linseed rich-diet). Four cows were selected from a dairy farm after a switch from a control diet to a linseed-rich diet. Among them, 2 cows (R-MFD) were resistant to MFD showing high milk fat content in both control (CTR) (4.06%) and linseed-rich diet (LIN) (4.36%); and 2 cows (S-MFD) were sensitive to MFD decreasing milk fat content after the change into the LIN diet (3.56 to 2.54%). Fresh milk samples were collected from each cow the week before and 2 weeks after the diet change. Transcriptome analysis was performed using RNA-sequencing technology with a HiSeq2000 platform. Differential expression analysis between cows S-MFD vs. R-MFD allowed to detect a large number of genes differentially expressed (DE) in both diets, CTR (n = 1,111) and LIN (n = 1,669). In addition, 726 genes were differentially expressed between CTR and LIN diets in cows R-MFD whereas only 38 genes were identified in cows S-MFD. Results showed an overexpression in genes and pathways related to fatty acid synthesis and lipid metabolism such as TR/RXR and PPARα/RXRα Activation pathways in cows R-MFD (CTR vs. LIN). Several genes and transcription factors such as FOXO3, MTOR, PPARA, PPARG, SREBF1 and MYC were identified acting as key regulators in R-MFD cows with linseed-rich diet. These results suggest that R-MFD cows could be activating a compensatory mechanism to increase the fatty acid synthesis in linseed-rich diets. It would be interesting to combine these results with the study of the structural variation in the whole transcriptome of S-MFD and R-MFD cows to contribute to the better understanding of molecular mechanisms affecting the MFD in cows.