The composition of grass/clover silage varies depending on time of harvest time. In particular silage from late regrowths is expected to contain lower fibre and higher linolenic acid concentrations compared to spring growth, thereby autumn silage is expected to increase linolenic acid content of milk fat. Rapeseed supplementation is expected to increase milk production and to increase all C18 fatty acids in milk fat. An interaction between rapeseed and silage type is expected, as hydrogenation of unsaturated fatty acids in rapeseed is expected to be less when low fibre silage is fed. Thirty-six Jersey cows were used in a 4 × 4 Latin square design, for 4 periods of 3 weeks and with a 2 × 2 factorial arrangement of treatments: spring grass/clover silage from primary growth or autumn grass/clover silage which was an equal mixture of 3rd regrowth and 4th regrowth, with or without rapeseed supplementation. Dry matter intake and milk production was higher for autumn than for spring silage. Rapeseed supplementation did not affect dry matter intake, but increased milk production. The concentrations of C18 : 1cis9, C18 : 2n6 and β-carotene and C18 : 3n3 in milk were increased whereas the concentrations of C16 : 0, riboflavin and α-tocopherol were decreased with autumn silage. The majority of C18 FAs in milk and α-tocopherol concentration increased with rapeseed whereas C11 : 0 to C16 : 0 FA were reduced. Autumn silage reduced biohydrogenation of C18 : 2n6, whereas rapeseed increased biohydrogenation of C18 : 2n6 and reduced biohydrogenation of C18 : 3n3. Apparent recovery of C18 : 2n6 was reduced with rapeseed. Minor interaction effects of silage type and rapeseed addition were observed for some milk fatty acids. Feeding silage from late regrowth increased linolenic acid concentration in milk fat. Rapeseed inclusion increased milk production, and increased C18 : 0 as well as C18 : 1 fatty acids, but not C18 : 2 and C18 : 3 in milk fat. Interactions between silage type and rapeseed supplementation were minimal.