Increasing milk omega-3 fatty acids (FA) content is desirable for human health. Our objective was to study the effect of 2 supplements rich in omega-3 in interaction with the proportion of concentrate in the diet on milk FA composition. The 2 sources of omega 3 were extruded flaxseed (FLAX, 1 kg.d−1) and alfalfa protein concentrate (APC, 2 kg.d−1) supplying respectively 115 and 49 g.d−1 omega-3 per cow. The cows were fed a corn silage based diet with 30% (C0) or 65% (C+) cereal based concentrate. The trial was carried out according a nested reversed design using 24 dairy cows averaging 117 ± 14 DIM with 2 periods of 14 d. Data were analyzed according a split plot design using proc mixed procedure. There was no significant interaction between the level of concentrate and the form of omega-3. C+ largely decreased milk fat content (−1.15 %) and yield (−301 g.d−1), the proportion of saturated FA (especially C16:0, C18:0, C4:0, C6:0 and C8:0) and increased trans C18:1, (especially t9, t10 and t12). Compared with FLAX, APC increased milk fat content (0.33 %), saturated FA (especially C4 to C12, C14:0 and C16:0) and decreased cis and all trans C18:1 isomers. Actually, FLAX induced high levels of cis and trans C18:1 isomers, c9t11CLA and C18:2 isomers. Transfer rate of C18:3 from feed to milk was much higher for APC (15.3 vs 4.7%) and increased with C+ when FLAX was fed. Decrease in milk fat content with C+ is classical and is due to the large amount of concentrate that had probably increased rumen propionic acid and t10 FA. It seems that these nutrients could have had a negative additive effect on milk fat content and yield. Especially, t10 FA could have inhibited FA synthesis in mammary gland. Decrease in milk fat content with FLAX is a consequence of heat seed treatment that increases oil release in the rumen, limiting ruminal biohydrogenation and inducing more trans FA. Positive effect of APC on transfer rate could be due to its manufacturing process inducing a lipid-proteic coagulum protecting FA. The effect of nature and protection of lipids on biohydrogenation depends on level of concentrates.