The aim of this study was to evaluate the effect of feeding oil supplement on mammary gland development and milk production responses in Holstein cows. Ten multiparous Holstein cows (42.2±9.2 d before calving, 3.25±0.25 body condition score, and 620±35 kg body weight) were randomly assigned to treatments. Treatments were a diet with oil added as palm oil (PO; n=5), or fish oil (FO; n=5) given to cows until 63 d in milk. Milk yield was recorded daily, milk composition (fat, protein, lactose, total solid and somatic cell count) was measured weekly and fatty acid profiles of milk fat were determined at first and last week of the experiment. Samples of mammary tissue were obtained at 7 and 63 d in milk by biopsy gun. Tissue slides were analyzed by Image J software. Results showed that fish oil supplemented diet compared to the palm oil supplemented diet increased milk production after 6 weeks of lactation (P<0.05), content of polyunsaturated fatty acids milk fat (P<0.05) and docosahexaenoic acid (P<0.01). Moreover, n-6:n-3 ratio was decreased by fish oil supplement (P<0.05). Histological studies showed that FO increased the relative percentage of tissue area occupied by epithelial cells as well as a number of total alveoli in each microscopic field (P<0.05). Data suggested that feeding fish oil during the dry period and early lactation could improve development and function of the mammary gland in the dairy cow.