Diets rich in unsaturated fatty acids (UFA) have been shown to increase the contents of some milk beneficial fatty acids (FA) including conjugated linoleic acid (CLA). The mechanism by which UFAs modulate cow’s genetics resulting in increased milk CLA content is not clear. This study examined the transcriptome of the bovine mammary gland following dietary supplementation with linseed oil. Twelve Holstein cows (35±10 kg milk; 150±50 DIM) were used in a complete randomized block design and fed a total mixed ration (control diet) for 28 d (d 1-28; control period, CP) followed by a treatment period (TP; d 29-56) consisting of control diet + 5% linseed oil (57% α-linolenic acid). Milk samples were collected weekly for fat, protein and individual FA determination. Mammary gland biopsies were performed on 6 cows on d 14 (CP) and d 35 and 56 (TP). Global transcriptome was analyzed using RNA-sequencing. Milk fat percent decreased (P < 0.0001) from 3.62% (CP) to 2.52% (TP) while protein percent was unchanged by treatment. The proportions of C4:0, C8:0, C14:0, C16:0 and C14:1 decreased (P ≤ 0.0003) while C18:1n-11t, C20:3n-3, C20:5n-3, C22:5n-3, CLA10t12c and CLA9c11t increased (P ≤ 0.035) during the TP. RNA-sequencing generated 459 million reads out of which 89% mapped to unique positions on the bovine genome. Eight genes (CSN2, CSN1S1, CSN1S2, LGB, CSN3, LALBA, COX1, and GLYCAM1) out of 11121 expressed constituted 73.70% of mapped reads. One hundred seventy-nine genes were significantly regulated (79 up- and 100 down-regulated) after FDR correction. Most affected genes were FBP2 (11-fold up-regulated) and MROH2B (4-fold down-regulated). Further regulated genes with roles in FA synthesis/uptake included LPIN1, SREBF1, INSIG1, FASN, BDH1, ACSS1, ACADVL, SLC25A34, SLC39A11, TIEG2, and CYP2B6. Differentially expressed genes were significantly enriched in several KEGG pathways including PPAR and insulin signaling pathways. This study has provided a broader picture of the transcriptomics events that are involved in mammary gland adaptation to a diet rich in UFA. Our study provides further knowledge on mammary lipogenesis and data that can be used to develop new nutritional strategies for a better management of milk increased beneficial FAs.