Health and nutrition professionals advise consumers to limit consumption of saturated FAs and increase consumption of foods rich in omega-3 FAs. We showed that feeding 3 kg of the flaxseed containing lipid supplement 12BT40 (N3Feed LLC; Tualatin, OR) increases omega-3 content in milk compared with its ground, unprocessed ingredients by protecting omega-3 FAs from ruminal biohydrogenation. To determine the optimal supplementation rate of 12BT40 for improving milk fatty acid profile without adversely affecting production parameters, we fed 6 mid- to late-lactation, pregnant Holstein cows (1 block each for primiparous and multiparous cows) for 6 weeks consecutively 0 (Control; 1 week), 0.91 (1 week), 1.81 (2 weeks), and 2.72 kg/d (2 weeks) 12BT40 as top-dressing. Milk and serum samples were collected at the end of each treatment period and analyzed for fatty acid profile and metabolic indicators, respectively. Individual feed intake and milk yield and components were measured to evaluate the effect of 12BT40 on production characteristics. Data were analyzed using PROC MIXED in SAS version 9.4. Fixed effects were supplementation rate (linear, quadratic and cubic) and parity; cow was the random effect. 12BT40 supplementation rates increased linearly (P_Linear < 0.0001) the omega-3 proportion in milk FAs (0.48, 1.92, 2.53, and 3.67 ± 0.30wt% for 0, 0.91, 1.81, and 2.72 kg/d of 12BT40) whereas the proportion of artherogenic 12:0, 14:0 and 16:0 was decreased (51.2, 41.1, 38.6, and 34.1 ± 1.0wt% for 0, 0.91, 1.81, and 2.72 kg/d of 12BT40; P_Linear < 0.0001). 12BT40 supplementation rates increased linearly (P_Linear = 0.009) milk yield (20.7, 20.9, 21.4, and 22.8 ± 1.3 kg/d for 0, 0.91, 1.81, and 2.72 kg/d of 12BT40) and had curvlinear relations with DMI (18.4, 17.5, 16.5, and 20.9 ± 0.6 kg/d for 0, 0.91, 1.81, and 2.72 kg/d of 12BT40) and milk fat content (3.96, 4.11, 4.69, and 4.06 ± 0.23% for 0, 0.91, 1.81, and 2.72 kg/d of 12BT40). Based on these results, we conclude that feeding up to 2.72 kg/d 12BT40 may improve milk fatty acid profiles without compromising production parameters.