The aim of the study was to assess the effects of partial replacement of soybean meal with a protein-equivalent amount of rapeseed cake in the diet on milking parameters and fatty acid (FA) composition of milk in dairy cows. Two groups of Holstein-Friesian cows, 8 each, consisting of randomised blocks were studied: a control group (C) was given a traditional high-protein supplement (extracted soybean meal) and the experimental group (E), had part of extracted soybean meal replaced with rapeseed cake. Dry matter intake and milk yield in both groups were not affected by the diet but milk fat percentage and yield were decreased in both groups. Rapeseed cake had no effect on milk acidity or on protein (including casein) and lactose contents. A lower concentration of urea in milk in E group indicated a proper ratio of protein to energy in the fodder. Health condition of mammary gland and indicators of metabolic profile were not affected by rapeseed cake supplementation. In E group, the share of atherogenic saturated fatty acids (FA) was reduced after 11 weeks: palmitic, by 26% and myristic, by 22%; moreover, as compared with control cows, the content of monounsaturated FA in milk increased by 44% after 3 weeks and by 68% after 11 weeks, t-18:1 and c-9 t-11 isomer of CLA increased about 2·5-fold after 11 weeks. In E group, the atherogenic index (AI) was significantly (P < 0·001) lower than in C (by 54% on average) and the decrease with time was considerable (by 29%, P < 0·001). Contents of odd- and branched- chain FA in milk were not significantly affected thus reflecting proper rumen function. Partial replacement of soybean meal with rapeseed cake in the diet of cows may improve both milking indices and FA profile of milk.