During CLA-induced milk fat depression (MFD) acetate and glucose are spared from milk fat synthesis and are available for other metabolic uses. Acetate is the major carbon source spared and although acetate deficiency does not cause milk fat depression the effect of acetate supply on lactation and the effect of spared acetate during MFD is not clear. The objective of this study was to compare the effect of CLA and acetate equivalent to that spared during MFD on milk production. Nine multiparous, lactating, ruminally cannulated Holstein cows (244 ± 107 DIM; mean ± SD) were randomly assigned to treatments in a 3 × 3 Latin square design. Experimental periods were 14 d in length and included 4 d for treatment and 10 d for washout period. Cows received the following treatments: control (CON), acetate [ACE; continuous infusion of 7 M/d acetate pH 6.1 (rumen)], or CLA [10 g/d trans-10,cis-12 CLA (abomasal)]. Milk samples were collected on the last 2 d of treatment for determination of fat, lactose and protein concentration, and milk fatty acid profile. Data were analyzed using the fit model procedure of JMP Pro. The model included the random effects of cow nested in sequence, sequence and period and the fixed effect of treatment. Dry matter intake, protein and lactose yield and percentage were not affected by treatments. Milk yield tended to be increased 11% in ACE (P = 0.09) compared with CON (22.8, 23.4 and 25.4 kg/d for CON, CLA and ACE, respectively). Milk fat yield increased 20% in ACE (P = 0.04) and decreased 23% in CLA (P < 0.02) and milk fat percent was decreased 30% by CLA (P < 0.001), but was not affected by ACE compared with CON. Concentration and yield of de novo FA (P < 0.001), while concentration of preformed FA (>C16) was increased by CLA (P < 0.001), compared with CON. Yield of de novo FA, palmitic acid and total C16 FA was increased by ACE (P < 0.05 for all) and concentration of palmitic acid was higher (P < 0.05) in ACE, compared with CON. In conclusion, acetate supply has an effect on milk production and milk fat synthesis, and spared acetate during MFD may improve energy status.