Unsaturated fatty acids (FA) and diet fermentability are key risk factors for milk fat depression. Linoleic acid (C18:2) is the greatest risk. Our hypothesis was that high oleic soybeans would result in higher milk fat yield compared with conventional soybeans. Eighteen non-cannulated multiparous Holstein cows (2.7 ± 0.8 parities; 153 ± 64 DIM; 45.7 ± 7.0 kg milk; 3.47 ± 0.58% milk fat; Mean ± SD at end of pretrial period) were used in a crossover design with 24 d periods. Each period consisted of 2 dietary phases. The first 14 d a diet with 5% roasted soybean in a 31% NDF diet was fed and the last 10 d a diet with 10% roasted soybeans and 28.5% NDF was fed. Treatments were conventional and high oleic roasted soybeans (Plenish, DuPont Pioneer). A common diet was fed for 7 d before the trial and during a 7 d washout between periods. Milk was sampled on d 10, 21, 27, and 31. Data were analyzed using the MIXED procedure of SAS (version 9.3, SAS Institute Inc., Cary, NC). The model included the random effects of period and cow and the fixed effect of soybean type, soybean level, and the interaction of soybean type and level. The preplanned contrasts tested the effect of treatment at each time point. There was no effect of high oleic acid soybeans on DMI or milk yield (averaged > 43 kg/d). Overall, high oleic soybeans increased milk fat percent 0.17 percentage units (P < 0.05) and tended to increase milk fat yield 89 g/d (P = 0.08) and there was no interaction of soybean type and level. The lower fiber and higher soybean diet increased milk fat concentration 0.2 percentage units and increased milk fat yield 92 g/d (both P = 0.01). There was no effect of high oleic soybeans on the concentration of de novo, 16 carbon, or preformed fatty acids in milk fat. The lower fiber and higher soybean diet decreased de novo synthesized FA and mixed source 16 C fatty acids by almost 3 and 9%, respectively, and increased preformed fatty acids over 10%. High oleic acid soybeans decreased trans-11 C18:1 and trans-10 C18:1 in milk. In conclusion, high oleic soybeans increased milk fat through decreased biohydrogenation induced milk fat depression. Additionally, increasing soybeans increased milk fat through providing preformed fatty acids.