Effects of grain type and dietary oil supplement on milk fat depression, milk fatty acid (FA) profile, and lactational performance of dairy cows were evaluated using 8 multiparous Holstein cows in a duplicated 4 × 4 Latin square design with a 2 × 2 factorial arrangement of treatments. 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 diets contained 28.5 and 31.2% of cereal grain in corn- and barley-based diets, respectively, as the sole source of grain. The forage component of the experimental diet was a mixture of corn silage (19.0% of DM) and alfalfa hay (21.0% of DM). Treatment periods were 25 d, with the final 7 d used for sample and data collection. Data were composited within period and subjected to MIXED MODEL procedure of SAS (SAS Institute, 2003) to account for effects of square, period within square, cow within square, treatments (grain type and oil supplement), and the interaction between grain type and oil supplement. Total FA intake was greater in corn-based diets and also in soybean oil supplemented diets (P ≤ 0.05). Fish oil decreased intake of all 18-carbon FA but increased intakes of other FA including C16:0, C20:5, and C22:6 (P ≤ 0.05). No significant differences existed in the DM intake and yield of milk or milk components between barley- and corn-based diets (P > 0.05). Fish oil negatively affected feed intake and yields of milk and milk components as compared with soybean oil (P ≤ 0.05). Although milk fat yield was not affected (P > 0.05), the barley-based diets increased the concentration and yield of medium-chain FA but decreased the concentration of long-chain FA as compared with corn-based diets (P ≤ 0.05). Corn-based diets increased concentration and yield of both trans-11 C18:1 and cis-9, trans-11 C18:2 which was a reflection of greater intake of cis-9, cis-12 C18:2 (25.1 g/d) as substrate for rumen biohydrogenation (P ≤ 0.05). Severity of MFD was greater for fish oil- vs. soybean oil which evidenced by the increased concentration and yield of biohydrogenation intermediates associated with MFD (especially trans-10 C18:1) in milk fat (P ≤ 0.05). However, fish oil increased concentration and yield of both trans-11 C18:1 and cis-9, trans-11 C18:2 as compared with soybean oil (P ≤ 0.05). Results indicated that there was no interaction between the type of grain and oil supplement on induction of MFD and milk fat yield. Although milk fat yield was not affected, milk fat composition was differently modified in barley- vs. corn-based diets. Observed lower milk fat yield for fish oil supplemented diets was coincided with the increased concentration and yield of biohydrogenation intermediates associated with MFD in milk fat.