Acetate is a major source of energy and substrate for milk fat synthesis in the dairy cow. We recently reported a linear increase in milk fat synthesis and greater than a 30% net transfer of acetate to milk fat with ruminal infusion of neutralized acetate. The objective of the current study was to investigate the ability of acetate mixed in a TMR to increase milk fat synthesis. Additionally, infusion of acetate results in an increase in plasma β-hydroxybutyrate so the effect of butyrate on milk fat was also investigated. Twelve multiparous lactating Holstein cows were randomly assigned to treatments in a 3 × 3 Latin square design with 14 d periods including 7 d of treatment and 7 d washout. Cows were fed ad libitum with a low risk diet for milk fat depression (33% NDF, 24% starch, 4.5% ether extract, 17.3% CP) and treatments were mixed into the basal diet. Treatments were on a dry matter basis: 3.1% NaHCO3 (control), 2.7% sodium acetate, and 2.4% calcium butyrate (carbon equivalent to acetate treatment). Data were analyzed by repeated measures and the model included the random effect of cow, period and sequence and the fixed effect of a covariate (d 0 of each period), treatment, time, and their interaction. Treatments were compared using a protected LSD. Feeding sodium acetate increased DMI by 2.7 kg (P < 0.05), had no effect on milk yield, and increased milk fat yield by 4.7% (P < 0.05) and concentration by 4.4% (P < 0.05) compared with control. Calcium butyrate decreased DMI by 2.3 kg, milk yield by 5%, milk fat yield by 8.8% and milk protein yield by 8.7% and concentration by 2.2%, compared with control (all P < 0.05). Sodium acetate increased concentration of 16 carbon mixed source fatty acids (FA) and myristic and palmitic acid (P < 0.05), while decreasing preformed FA, compared with control. Calcium butyrate had no effect on concentration of milk FA by source, but increased concentration of trans-10 C18:1 in milk by 18% (P < 0.05), indicating a shift in rumen biohydrogenation pathways. Our data demonstrates that milk fat yield can be increased by feeding sodium acetate and that butyrate does not increase milk fat at equivalent levels.