Thirty-six Holstein-Friesian crossbred lactating dairy cows were used to determine the effects of linseed oil supplementation on performance and milk fatty acid (FA) profile. Three treatments were as follows: basal diet (56:44 Roughage:concentrate [R:C] ratio, dry matter basis) supplemented with 500 g of palm oil as control (PO), 500 g mixture (1:1, w/w) of palm oil and linseed oil (POLSO) and 500 g of linseed oil (LSO). The LSO supplementation had no effects on total dry matter intake (DMI), milk yield and milk composition. Compared to control cows, cows supplemented with LSO increased milk concentrations of cis-9,trans-11 conjugated linoleic acid (CLA) and n-3 FA (P < 0.05), particularly C18:3n-3, C20:5n-3 and C22:6n-3. Feeding LSO reduced concentrations of milk short- and medium-chain saturated fatty acids (P < 0.05) while it increased concentration of milk unsaturated fatty acids (P < 0.05). Milk proportions of n-3 FA increased, whereas n-6/n-3 ratio decreased in the LSO as compared with the control (P < 0.05). In conclusion, supplementing dairy cows' diet based on corn silage with LSO at 500 g/day could improve the nutritional value of milk with potential health-beneficial FA without detrimental effect on milk composition or cow's performance.