The effect of barley grain processing and source of supplemental fat on performance of lactating dairy cows were studied in a replicated 4 × 4 Latin square design with 21-d periods and a 2 × 2 factorial arrangement. Eight Holstein cows with mean body weight (BW) of 572 ± 71 kg and 45 ± 10 days in milk were allocated to 4 dietary treatments including 1) ground barley with cottonseed 2) pelleted barley with cottonseed 3) ground barley with canola seed 4) pelleted barley with canola seed. The nitrogen intake and fecal N were higher in cows fed ground barley in comparison with those fed pelleted barley. Source of supplemental fat or barley processing had no effect on milk fat and milk protein contents. Milk SNF yield was higher in cows fed canola as supplemental fat source and ground barley (P < 0.05). Milk yield was affected by method of barley grain processing and was 0.64 to 1.9 kg/d higher in cows fed ground barley compared with those fed pelleted barley (P = 0.04). Plasma concentrations of glucose, NEFA, BHBA, cholesterol, triglycerides and blood urea nitrogen were similar in all treatments. Dry matter intake was affected by barley grain processing. The cows fed ground barley consuming 1.15 to 2.18 kg/d more DM compared with those fed pelleted barley (P = 0.04).Total tract digestibilities of DM, crude fat, ADF, NDF and OM were not affected by the barley grain processing as well as source of oilseed. The results indicated that interactions between barley grain processing and source of supplemental dietary fat can improve the performance of lactating dairy cows, However, more detailed studies are required