Abstract In this study, the effect of the lactation stage on changes in the fat and cholesterol content and fatty acid composition of colostrum and milk of Wielkopolska mares' was investigated. Colostrum and milk samples from 10 lactating mares were collected at the beginning of lactation, on the 1st and 2nd day after foaling and then, starting from the first month of lactation, at 4 weeks' intervals up to the fifth month of lactation. Statistically significant differences were found between mares' colostrum and milk in the fat and cholesterol content as well as in the percentage rate of total fatty acids which occurred in significant quantities. Together with the lengthening of the lactation time, the content of fat and cholesterol in the mares' milk was observed to decrease. The authors also found a significant impact of the lactation period on changes in the ratio of polyenoic fatty acids from the n-6 to n-3 family. Bearing in mind the fat and cholesterol content and a more favourable (from the nutritional point of view) ratio of fatty acids from the family of n-6 to n-3, it is recommended to collect milk from mares in the 4th and 5th month of lactation.