Supplementing a high dose of dietary conjugated linoleic acid (CLA) inhibits milk fat synthesis in dairy cows immediately postpartum. During negative net energy balance (EBAL), it appears that moderate CLA-induced milk fat depression causes a positive response in milk yield; however, as milk fat depression becomes more severe, the milk yield response diminishes. Multiparous Holstein cows (n = 31) were randomly assigned to 1 of 3 treatments beginning 9 +/- 6 d before expected calving and ceased at 40 d in milk (DIM): 1) 578 g/d of a rumen-inert (RI) palm fatty acid distillate (control), 2) 600 g/d of RI-CLA for the entire trial period (CLA-1), and 3) 600 g/d of RI-CLA until 10 DIM followed by 200 g/d for the remainder of the trial (CLA-2). Each dose provided equal amounts of fatty acids by replacing and balancing each treatment with a RI palm fatty acid distillate. Doses provided a total of 522 g of fatty acids/ d and 0, 174, or 58 (depending upon DIM) g of CLA (mixed isomers)/d. To improve palatability, doses were mixed with 600 g/d of dried molasses; one-half of the supplement was fed at 0800 h, and the remainder at 1900 h. Individual milk yield, dry matter intake, and body weight were recorded daily and milk composition determined every other day. There was no overall CLA effect on either the content or yield of milk protein or lactose. Both CLA treatments decreased overall milk fat content (26.0 and 18.3%) and yield (22.5 and 17.3%) with CLA-induced milk fat depression becoming significant by d 8. The CLA-induced milk fat depression increased in magnitude with progressing DIM until reaching a plateau on d 18 for CLA-1 (43%) and on d 14 for CLA-2 (33%), although neither milk fat trans-10, cis-12 CLA content (1.8 mg/g) nor its transfer efficiency (6.3%) changed over time. Treatments had no effect on overall dry matter intake or milk yield, but there was a treatment x time interaction for milk production, as cows fed either CLA treatment had increased milk yield after the second week of lactation. Cows fed either CLA treatment had a significant improvement in overall EBAL (-5.1 vs. -1.8 Mcal/d), a decrease in nonesterified fatty acid levels (12%), and an increase in glucose levels (11%). A dietary supplement containing trans-10, cis-12 CLA markedly improves EBAL and bioenergetic variables and increases milk yield in the total mixed ration-fed transitioning dairy cow.