Fat supplements are commonly fed to increase dietary energy density and improve animal performance. However, metabolic effects may vary depending on fatty acid (FA) chain length. Eleven multiparous Holstein dairy cows (150 ± 52 DIM; Mean ± SD) were randomly assigned to treatment sequence in a replicated 3 × 3 Latin Square design testing the effect of saturated fat supplements differing in chain length on milk production and composition, and on milk FA profile. Treatment periods were 7 d in length and were separated by 7 d of washout. Treatments were administered as abomasal infusions of emulsions providing 280 g/d of FA from: 1) free FA enriched in palmitic acid (>85% 16:0; PA), 2) free FA enriched in stearic acid (98% 18:0; SA), or 3) medium chain triglycerides (50% 8:0 and 50% 10:0; MCT),. Milk yield was recorded and samples taken on the last 3 d of each period. The statistical model included the random effects of period, cow and sequence, and the fixed effect of treatment. Preplanned contrasts tested were PA vs. SA, and PA vs. MCT. Fat- and energy-corrected milk were not affected by treatments, and averaged 36.0 ± 2.0 kg/d and 35.1 ± 2.0 kg/d, respectively (P > 0.11). Milk fat content was higher in PA (4.49%) than in SA (4.18%, P < 0.001), but was not different between PA and MCT (4.37, P = 0.17). Similarly, milk fat yield was increased by PA (1.56 kg/d) relative to SA (1.46 kg/d, P = 0.01) and tended to be higher in PA than in MCT (1.48 kg/d; P = 0.06). De novo synthesized FA were higher in both SA and MCT (27.3 and 30.5% of FA, respectively) compared with PA (24.5% of FA; P < 0.001). The sum of 16:0 and cis-9 16:1 was higher in PA (43.7% of FA) relative to MCT and SA (38.0 and 35.6, respectively; P < 0.001). Lastly, the concentration of 18:0 plus cis-9 18:1 was increased by SA (31.2% of FA) relative to PA (26.6% of FA; P < 0.001), but was not different between PA and MCT (25.8%, P = 0.42). The chain length of fat supplements infused into the abomasum affected milk fat and fatty acid profile.