We recently reported that supplementation of 2-hydroxy-4-(methylthio)butanoate(HMTBa) reduced the shift to the alternate biohydrogenation pathway and maintained higher milk fat yield in high producing cows fed diets lower in fiber and higher in unsaturated fatty acids (FA). The objective of this experiment was to verify the effect of HMTBa (Alimet, Novus International, Inc., St. Charles, MO) on biohydrogenation intermediates and milk fat synthesis. Twenty-two rumen cannulated high-producing Holstein cows [168 ± 66 DIM; 42 ± 7 kg milk/d (Mean ± SD)] were used in a randomized design performed in 2 blocks (1 = 14 cows, 2 = 8 cows). Treatments were control (corn carrier) and HMTBa (0.1% of diet DM). The experiment included a 7-d covariate period followed by 3 phases that fed diets with increasing risk of milk fat depression (MFD). The diet during the covariate and low-risk phase (7 d) was 32% NDF with no additional oil. The diet during the moderate-risk phase (17 d) was 29% NDF with 0.75% soybean oil. Soybean oil was increased to 1.5% for the last 4 d. Milk yield and DMI were measured daily. Milk was sampled every 7 d and analyzed for fat, protein and FA profile. Data were analyzed using PROC Mixed with repeated measures and the effect of treatment was tested at each time point. There was no effect of block or interaction of block and other fixed effects. There was no overall effect of treatment or treatment by time interaction for DMI (P = 0.4), milk yield (P = 0.4), and milk protein concentration (P = 0.9) and yield (P = 0.6). There was an effect of treatment, but no treatment by dietary phase interaction on milk fat with HMTBa increasing milk fat percent (3.2 vs. 3.6%, P < 0.01) and yield (1342 vs. 1543 g/d, P = 0.02). Additionally, HMTBa decreased the concentration (1.29 vs. 0.81 g/100 g of total FA, P = 0.02) and yield (14.7 vs. 10.9 g/d, P = 0.01) of trans-10 18:1 in milk across the entire feeding period. In conclusion, HMTBa prevented the increase in trans FA associated with MFD and maintained milk fat yield when cows were fed a diet with moderate risk of diet-induced MFD.