Effects of grain type and dietary oil supplement on milk fat depression and milk fatty acid (FA) composition of dairy cows were evaluated using eight multiparous Holstein cows (77 +/- 22.1 days in milk; mean +/- SD) in a replicated 4x4 Latin square design with a 2x2 factorial arrangement of diets. Experimental diets contained either ground barley or ground corn supplemented with either fish oil or soybean oil at 2% of dietary dry matter (DM). Experimental periods were 25 d, with the final 7 d used for sample and data collection. Dry matter intake tended (P=0.09) to be greater for barley- vs. corn-based diets (23.2 vs. 22.3 kg/d), but was reduced for the fish oil compared to soybean oil supplemented diets (21.1 vs. 24.3 kg/d; P < 0.001). Total FA intake was greater in corn-based diets and also in soybean oil supplemented diets. Regardless of type of the diet fed, MFD occurred. Although milk fat yield was not affected, the barley-based diets increased (P < 0.001) the concentration of mixed-origin FA (C16:0 plus cis-9 C16:1) but decreased the concentration of preformed FA (P < 0.001) as compared with corn-based diets. Corn-based diets increased concentration of both trans-11 C18:1 (P=0.03) and cis-9, trans-11 C18:2 (P=0.01) which was a reflection of greater intake of cis-9, cis-12 C18:2 as substrate for rumen biohydrogenation. Severity of MFD was greater for fish oil than for soybean oil which was evidenced by the increased concentration and yield of biohydrogenation intermediates (especially trans-10 C18:1) associated with MFD (r =-0.61; n =32) in milk fat. However, fish oil increased concentration of both trans-11 C18:1 (P=0.001) and cis-9, trans-11 C18:2 (P < 0.001) as compared with soybean oil. Grain type and oil supplement did not interact to affect milk odd- and branched-chain FA (OBCFA) concentration. Cows fed barley-based diets (P < 0.001) and soybean oil supplemented diets (P < 0.001) produced greater OBCFA in milk fat. Overall, there was no interaction between the type of grain and oil supplement on induction of MFD and milk fat yield. However, milk fatty acid composition was differently modified by the grain type and an increase in the concentration of mixed-origin FA of fish oil supplemented diets was unable to attenuate the severity of MFD because of the concomitant decrease in concentrations of de novo synthesized- and preformed-FA as compared with soybean oil supplemented diets.