5×5 Latin-square experiment (21-d periods) was conducted to measure the effects of replacing of barley by sorghum grain on milk yield, milk composition, feed efficiency, dry matter intake (DMI), body weight and apparent digestibility of organic matter (OM). Five primiparous and five multiparous (n=10) lactating Holstein cows, 85±10 d in milk were given a total mixed ration of 46: 54 forage: grain ratio (DM basis). Sorghum grain substituted barley in ratios of 0: 100, 25:75, 50:50, 75:25 and 100:0 in diets 1, 2, 3, 4 and 5, respectively. The results showed significant differences (P<0.05) in many parameters in response to increasing sorghum levels. Diet 5 showed higher DMI (20.3 vs. 19.4 kg d-1), daily milk yield (25.8 vs. 24.8 kg d-1), production of 3.2 % FCM (27.4 vs. 24.7 kg d-1), milk fat (3.58 vs. 3.02%), fat yield (0.92 vs. 0.75 kg d-1), milk protein (3.0 vs. 2.7%), protein yield (0.75 vs. 0.67 kg d-1), milk lactose (4.93 vs. 4.47%) and milk solids-not-fat (8.58 vs. 8.17%), as compared with diet 1. However, feed efficiency, body weight, and apparent digestibility of organic matter were not affected by any of the treatments (P>0.05). It is concluded that substitution of sorghum grain for barley may manipulate rumen metabolism and improves milk composition.