The effects of dietary starch fermentability on plasma metabolites and hormones, milk production, and milk fatty acid profile were evaluated in a crossover study. Thirty-two multiparous Holstein cows (121 +/- 48 d in milk, 41 +/- 9 kg/d 3.5% fat-corrected milk [FCM]; mean +/- SD) were randomly assigned to treatment sequence and were fed a diet intermediate to the treatments during an initial 21-d period. Treatments were dry ground corn grain (DG) and high moisture corn (HM) harvested from the same field. Treatment periods were 14 d, with the final 4 d used for data and sample collection. Diets included corn silage and alfalfa haylage at a 21 ratio and were ~26% neutral detergent fiber, 16.5% crude protein, 32% starch, and 3.5% fatty acids. High moisture corn increased plasma glucose, nonesterified fatty acid, and triglyceride concentrations, but treatment had no consistent effect on yield of milk or FCM. High moisture corn increased trans C(181) concentrations at an increasing rate as production level decreased across cows, and milk fat depression was evident in cows below approximately 40 kg/d FCM yield. In contrast, production level had little influence on milk trans C(181) concentration for DG. Milk trans C(181) concentration was negatively correlated with milk fat concentration, as was trans-9 C(181), trans-10 C(181), and cis-9, trans-11 C(182). Concentration of trans-10, cis-12 C(182) was not correlated with milk fat concentration. Production level may influence biohydrogenation patterns and trans C(181) production because of differences in rumen environment; rumen pH and dilution rate can alter metabolism and populations of rumen microbes. Diets with highly fermentable starch sources and without supplemental dietary polyunsaturated fatty acids can induce milk fat depression in lower-producing cows, likely because of increased production of trans C(181) fatty acids.