We evaluated the effects of abomasal infusion of emulsifiers on fatty acid (FA) digestibility and production responses of lactating dairy cows. Eight rumen-cannulated multiparous cows (89 ± 13 DIM) were assigned to a treatment sequence in 4 × 4 Latin squares with 18 d periods consisting of 7 d of washout and 11 d of infusion. Treatments were abomasal infusions of water carrier only (CON) or 30 g/d of different emulsifiers enriched in different FA: polysorbate-C16:0 (Tween-40; T40), polysorbate-C18:0 (Tween-60; T60), and polysorbate-C18:1 (Tween-80; T80). Emulsifiers were dissolved in water and delivered at 6 h intervals. Cows were fed the same diet which contained (% DM) 32% NDF, 16% CP, 26% starch, and 3.4% FA (2% DM from a FA supplement containing 34% C16:0 and 47% C18:0). The statistical model included the random effect of cow within square and the fixed effects of treatment, period, square, and their interactions. Pre-planned contrasts included CON vs. average of T40, T60, and T80 (TWEEN); CON vs. T80; and T80 vs. the average of T40 and T60 (T40+T60). Compared with CON, TWEEN did not affect DMI or digestibility (P > 0.15), but increased milk fat content (P = 0.04) and tended to increase milk fat yield (P = 0.06) and 3.5% FCM (P = 0.09). Compared with CON, T80 increased total (73.3% vs. 69.3%, P = 0.02), 16-carbon (73.1% vs. 69.8%, P = 0.04), and 18-carbon (73.0% vs. 68.8%, P = 0.02) FA digestibility. T80 tended to decrease DMI (P = 0.08) and increase digestibility of DM (P = 0.09) and NDF (P = 0.09). T80 increased milk fat content (3.70% vs. 3.33%, P = 0.04) and yield (1.66 vs. 1.49 kg/d, P = 0.04), tended to increase 3.5% FCM (P = 0.06), and tended to reduce milk protein yield (P = 0.06) compared with CON. Compared with T40+T60, T80 increased total (73.3% vs. 69.4%, P = 0.01), 16-carbon (73.1% vs. 70.1%, P = 0.04), and 18-carbon (73.0% vs. 68.7%, P = 0.01) FA digestibility and tended to increase DM digestibility (P = 0.06), but did not affect DMI (P = 0.19) or NDF digestibility (P = 0.29). In conclusion, T80 infusion increased the digestibility of total, 16-carbon, and 18-carbon FA, compared with control and T40+T60, suggesting that the predominant FA attached to polysorbate affects its ability to improve FA digestibility.