Effects of grain type and dietary oil supplement on production performance, energy balance, metabolic heat production, and markers of liver function of heat-loaded lactating dairy cows were evaluated using 8 multiparous Holstein cows (77.0 d in milk) in a duplicated 4 x 4 Latin square design with a 2 x 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. Mean daily maximum temperature, minimum relative humidity, and maximum temperature humidity index were 35.3 degrees C, 11.3%, and 77.0, respectively. Dietary treatment did not affect rectal temperature (38.9 degrees C), but respiration rate tended to decrease in cows fed fish oil versus soybean oil. Dry matter intake decreased for the fish oil supplemented diets (21.1 vs. 24.3 kg/d), which was negatively correlated with plasma concentrations of alkaline phosphatase (r = -0.45; n = 32) and malondialdehyde (r = -0.26; n = 32). Actual milk yield (41.9 kg/d) and energy-corrected milk yield (36.6 kg/d) were not affected by grain type, whereas feeding fish oil decreased milk yield as compared with soybean oil (40.4 vs. 43.4 kg/d). Milk fat depression occurred in all dietary treatments, especially when cows were fed fish oil because of the presence of polyunsaturated FA in the diets. trans-10 C18:1 was negatively correlated with milk fat yield (r = -0.38; n = 32). Daily milk cis-9,trans-11 C18:2 secretion was 29.6% less in cows fed barley- versus corn-based diets but 31.8% greater in cows fed fish oil as compared with cows fed soybean oil. Because of a lower dry matter intake, metabolic heat production was decreased in cows fed fish oil relative to cows fed soybean oil. Although feeding fish oil versus soybean oil decreased net energy for both maintenance and lactation, net energy balance remained unchanged across treatments. In vivo plasma lipoperoxidation was greater in cows fed fish oil versus soybean oil, which substantiated increased susceptibility of plasma lipoperoxidation when cows were fed fish oil. Plasma concentration of malondialdehyde was positively correlated with plasma aspartate aminotransferase (r = -0.38; n = 32), which is an indicator of liver function in heat-loaded cows. Results suggest that in heat-loaded cows fed diets supplemented with soybean oil versus fish oil, biosynthesis in the mammary gland was prioritized over anabolism and oxidation in peripheral adipose and muscle tissues regardless of type of grain used.