The objective of this study was to compare the effect of contrasting sources of dietary n-6 and n-3 PUFA on expression of genes related to lipid metabolism in dairy cows. During 63 days, fifteen lactating cows were assigned to a control or basal diet containing no added lipid (n = 5 cows); and treatment diets supplemented with SO (n = 5 cows; unrefined soybean oil; 2.9% of DM) or FO (n = 5 cows; fish oil manufactured from salmon oil; 2.9% of DM). Plasma for fatty acid (FA) analysis and milk somatic cells (MSC) were obtained from all cows at the beginning of the study (day 0) and on days 21, 42 and 63. Plasma was used to determine FA transport dynamics. Compared with control and FO, plasma from SO had increased contents of C18:1 cis-9, C18:1 trans-11, C18:2 cis-9, trans-11 and total monounsaturated FA. On the other hand, compared with control and SO, FO increased plasma contents of C20:3 n-3, C20:3 n-6, C20:4 n-6, C20:5 n-3, C22:6 n-3 and total polyunsaturated FA. Moreover, plasma C18:3 n-3 and C20:5 n-3 increased over time for all diets. Compared with control, SO downregulated ACACA, INSIG1, and DGAT1, whereas FO downregulated ACACA, PPARGC1, LPIN1 and FABP3 on day 63, in MSC. At different time-points, SO and FO downregulated genes related to synthesis and intracellular transport of FA, synthesis of triglycerides, and transcription factors.