Our objective was to observe the effects of milk fat substrates (MFS) [acetate combined with long-chain fatty acid (FA)] supplementation on lactation performance and milk FA composition of cows fed an energy- and nutrient-deficient diet. Eight Holstein cows (99 ± 11 d) were averagely assigned to two diets based on either corn stover (CS) or mixed forages (MFs). Experiment included two stages and each lasted for 27 d with a 21 d preliminary and a 6 d infusion. Cows with CS diet [NE_L = 1.35 Mcal kg^-1 of dry matter (DM)] received carrier at the first 3 d and MFS at the following 3 d of infusion period. Cows with MF diet (NE_L = 1.66 Mcal kg^-1 of DM) received carrier during the 6 d infusion period. The differences in milk production, milk yields of fat, and protein between the two diet groups were compensated by MFS infusion (from -3.5 to -1.3, -0.19 to -0.08, and -0.13 to -0.07 kg d^-1, respectively). Infusion of MFS increased the concentrations of 18:2 and 18:3 in milk fat. The results suggested that supplement of MFS could influence milk fat synthesis and lactation performance in cows fed an energy- and nutrient-deficient diet.