The objective of this study was to determine the effects of intravenous infusion of olive oil, safflower oil and flaxseed oil in various combinations on milk fat composition in lactating dairy cows. Eight Chinese Holstein cows (101 ± 11 DIM) were used in a replicated 4 × 4 Latin square design. Each period lasted 12 d with 5 d of infusion and 7 d of adaptation. Treatments were jugular vein infusion of 1) 170 g/d of oil mixture contained olive and safflower oil (43.10% cis-9 C18:1, 42.58% cis-6 C18:2 and 1.08% n-3 C18:3, OS); 2) 167 g/d of oil mixture contained olive and flaxseed oil (43.69% cis-9 C18:1, 13.89% cis-6 C18:2 and 29.94% n-3 C18:3, OF); 3) 161 g/d of oil mixture contained safflower and flaxseed oil (15.12% cis-9 C18:1, 44.72% cis-6 C18:2 and 30.50% n-3 C18:3, SF); and 4) 224 g/d of oil mixture contained olive, safflower and flaxseed oil (33.02% cis-9 C18:1, 32.94% cis-6 C18:2 and 22.63% n-3 C18:3, OSF). Treatment emulsions were consisted of water, oil mixture, 10 g/L of soy lecithin and 25 g/L of glycerol. The volume of the emulsion was 2 L/d per cow. Infusion process lasted for 6 h per day. Milk samples were collected twice daily during the last 2 d of each 5 d infusion period. Data were analyzed by using the Mixed procedure of SAS. Significance was declared at P < 0.05. Milk fatty acid from OF had greater concentration of cis-9 C18:1 (26.29%) compared with 21.80% from SF and 22.25% from OSF (SEM = 1.04). The concentration of cis-6 C18:2 from OF (4.36%) had the lowest level (P < 0.0001) compared with 6.77%, 7.07% and 7.00% from OS, SF and OSF (SEM = 0.21), respectively. The concentration of n-3 C18:3 in OS (0.66%) was lower (P < 0.0001) than in OF (3.57%), SF (3.26%) and OSF (3.34%), SEM = 0.13. The concentrations of C18:0 and short- and medium-chain fatty acids were not changed through treatments, whereas C16:0 was lower (P = 0.0202) in treatment OF (30.70%) than in treatment OS (32.62%), SEM = 0.74. Infusion of different oil combinations increased target fatty acids in milk and markedly altered milk fat composition.