To determine the effect of dietary supplementation with vegetable oilseeds on the composition of bovine milk fatty acids(FAs), 40 Holstein dairy cows were used with a complete randomized design. At the beginning of the experiment, the cows were 150±25 day in milk (DIM). Total duration of the experiment was six weeks. Measurements were made during the last three weeks. Cows in four treatments were fed with a basal diet (CT) or basal diet supplemented with either whole full fat soybean (WFS), full fat expanded soybean (FPS) or whole full fat soybean with whole cottonseed and full fat expanded soybean (MIX). The composition of the milk fat was analyzed by gas chromatography. Relative to control, the conjugated linoleic acid (CLA) concentration in milk fat from cows on FPS was significantly increased by 83.88% (P＜0.05). The proportions of C12:0 were decreased by 35.7, 35.51, and 38.65% in milk fat from cows on WFS, MIX, and FPS compared with cows on CT. Similar decreases in C14:0 were 23.83, 24.85, and 31.48% in WFS, MIX, and FPS treatments, respectively.Feeding vegetable oilseeds increased the proportion of healthy FAs (mainly CLA), whereas decreased the concentration of C12:0 and C14:0. Therefore, milk and dairy products would have higher nutritive and therapeutic value.