Studies on nutritional regulation of digestive enzymes in ruminants are scarce. Fat supplementation of diets for dairy cows changes the supply of nutrients for absorption and transport. The aim of this experiment was to study the effect of the physical form of rapeseed (Brassica napus) fat on the duodenal enzyme profile in dairy cows. Four mid-lactation Danish Holstein dairy cows were submitted to an experiment with a 4 × 4 Latin square design and fed four rations containing 50% forage and 50% concentrate on a dry matter basis. The four rations were: a control ration with rapeseed meal (low fat) and three fat-supplemented rations with either rapeseed cake (RSC), whole cracked rape seed (WCR), or rapeseed oil (RSO). The correlation coefficients among duodenal enzyme activities and the relationship between α-amylase and protease activities were examined. Diurnal samples were taken from the duodenum, frozen immediately and stored frozen (–20°C) before analyses. Additional fat increased the soluble protein content in duodenal chyme (P < 0.05). Neither α-amylase nor the protease or soluble sugar concentrations were affected by the treatments. Lipase activity increased for RSO compared with the control. The relationship between α-amylase and protease activities showed a parabolic behaviour (r = 0.77; P = 0.061). In conclusion, inclusion of rapeseed oil in the ration of dairy cows under the experimental conditions increased the soluble protein concentration and lipase activity in duodenal chyme.