We evaluated the effects of altering the dietary ratio of palmitic (C16:0) and oleic (cis-9 C18:1) acids on production responses of cows with a wide range of milk production (32 to 65 kg/d) in a crossover design experiment with a covariate period. Thirty-two multiparous Holstein cows (144 ± 94 DIM) were assigned randomly within level of milk yield to treatment sequence. Treatments were diets supplemented with FA blends (1.5% of diet DM) that provided 80% C16:0 + 10% C18:1 (PA) and 60% C16:0 + 30% C18:1 (PA+OA). The corn silage and alfalfa-based diets contained 20.0% forage NDF, 28.5% starch and 17.1% CP. Treatment periods were 21 d with the final 5 d used for data and sample collection. The statistical model included the random effect of cow, the fixed effect of treatment, period, preliminary milk yield (PMY), and 2-way interactions. Linear effects for the interaction between PMY and treatments were added to evaluate responses to treatment by level of milk yield. There were no effects of treatments on DMI (P = 0.34), milk yield (P = 0.38), ECM (P = 0.35), BW (P = 0.74), or BW change (P = 0.54). Compared with PA+OA, PA increased fat yield (1.92 vs. 1.83 kg/d, P < 0.01) and protein yield (1.61 vs. 1.55 kg/d, P = 0.03). PA also increased the yield of de novo (448 vs. 428 g/d, P < 0.05) and mixed (749 vs. 669 g/d, P < 0.01) milk FA and decreased the yield of preformed FA (605 vs. 627 g/d, P < 0.05) compared with PA+OA. Interactions were detected between treatment and PMY for DMI and ECM (linear interaction both P < 0.05), and a tendency for milk yield (linear interaction P = 0.12); lower-producing cows (less than 45 kg/d) had increased DMI and ECM on the PA diet whereas higher-producing cows (over 55 kg/d) had increased DMI and ECM on PA+OA. A linear interaction was also detected between treatment and PMY for mixed milk FA yield (linear interaction both P < 0.10) and a tendency for de novo milk FA yield (linear interaction P < 0.15). Our results demonstrate that production responses (DMI, milk yield, and ECM) of high-producing cows was better with a fat supplement containing more C18:1, while lower-producing cows responded better to a supplement containing more C16:0.