To evaluate the interaction between the levels of dietary concentrate and linseed oil (LO) on milk fatty acid (FA) profile, 24 Holstein cows were used in a randomised complete block design based on days in milk, with a 2×2 factorial arrangement of treatments. Within each block, cows were fed one of four experimental diets containing 30% concentrate (LC) or 70% concentrate (HC), without LO (NLO) or with LO supplemented at 3% of dietary dry matter. Milk FA profiles were analysed with a special emphasis on the intermediates of the predominant trans-11, and a putative trans-13 pathways of ruminal biohydrogenation of cis-9, cis-12, cis-15 18:3. Feeding LO increased the concentrations of cis-9, cis-12, cis-15 18:3 and trans-11, cis-15 18:2 in milk fat, and these increases were of a higher magnitude when LO was added in HC as compared with LC diet (interaction of LO by concentrate). A treatment interaction was also observed for the level of trans-11 18:1 which was higher when feeding LO, but for which the increase was more pronounced with the LC as compared with HC diet. The concentrations of cis-15 18:1 and cis-9, trans-11, cis-15 18:3 were higher in cows fed LO, but feeding HC diets decreased milk fat content of cis-15 18:1 and a tendency for a decrease in cis-9, trans-11, cis-15 18:3 was apparent. Feeding LO increased milk fat content of trans-13 18:1 and cis-9, trans-13 18:2, while the concentrations of these two isomers were not affected by the level of dietary concentrates. The isomer cis-9, trans-13, cis-15 18:3 has not been detected in any of the milk samples. In conclusion, interactions were observed between LO and dietary concentrates on the proportions of some intermediates of the trans-11 biohydrogenation pathway. The presence of trans-13 18:1 and cis-9, trans-13 18:2 supports the existence of a trans-13 pathway, but an 18:3 intermediate with a trans-13 double bond has not been identified.