Animal responses to dietary treatment may interact with metabolic state, which differs for cows across a wide range of milk yield. Responses to dietary saturated vs. unsaturated fatty acid (FA) supplement was evaluated using 32 multiparous Holstein cows arranged in a crossover design with 14-d periods. Treatments were 2.5% FA from unsaturated FA (calcium salts of palm FA) or saturated FA (prilled, hydrogenated free FA). Unsaturated FA treatments decreased dry matter intake (0.8 kg/d) and time spent ruminating (25 min/d) compared with saturated FA treatment. Treatments did not differ in milk or 3.5% fat-corrected milk yield. Intake and milk yield responses were not related to milk yield across cows. Saturated FA treatment increased milk protein and lactose concentrations, but treatment did not affect yield of milk components. Saturated FA treatment increased insulin over 25% and decreased nonesterified FA nearly 20% with no effect on plasma somatotropin, glucose, or beta-hydroxybutyrate concentrations. Milk protein concentration and yield responses to treatment were positively correlated with pretrial fat-corrected milk yield. Milk protein response was not related to insulin response, supporting the importance of insulin sensitivity in control of milk protein synthesis. Unsaturated FA treatment decreased dry matter intake and rumination time compared with saturated FA treatment, consistent with reports of unsaturated fat increasing satiety and decreasing gut motility. Decreased milk protein synthesis by fat supplementation may be related to FA saturation and milk yield of cows.