The objective of this research was to determine the effect of feeding fish oil, soybean oil, or their combination on milk fatty acid profiles, especially omega-3, omega-6 and omega-3/omega-6 ratio. Milk was collected from 20 primiparous Holstein cows that were distributed into four groups and arranged in a completely randomized design with 35 days period to determine the effect of feeding fish oil, soybean oil, or their combination on milk production and composition. Experimental diets consisted of: 1) control diet; 2) a diet with 3% (DM basis) added fat from menhaden fish oil; 3) a diet with 3% added fat from soybean oil and 4) a diet with 1.5% added fat from fish oil and 1.5% fat from soybean oil. Dry matter intake (18.47, 18.87, 18.33 and 18.63 kg day-1, for control, fish oil, soybean oil and combination diets, respectively) and milk production (30.31, 32.15, 31.19 and 31.59 kg day-1) were higher for cows that consumed 3% fish oil containing diet. Milk from cows fed control, fish oil, soybean oil and fish oil with soybean oil diets contained 3.45, 2.72, 2.96 and 2.87% fat, respectively. Concentration of total omega-3 fatty acids (0.87, 1.28, 0.96 and 1.18 g/100 g of fatty acids) in milk fat were higher for cows that consumed either fish oil-containing diet, especially the 3% fish oil diet. The n-6:n-3 ratio (4.57, 2.62, 6.17 and 4.08) in milk fat was lower for fish oil diet. These results showed that fish oil modifies fatty acids profile of milk fat and increased the proportion of beneficial fatty acids for human health.