The objective of the present study was to evaluate the impact of supplementing ewes with eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) during late gestation on ewe colostrum, and milk production and composition. Eighty-four gestating ewes (28 pens) were blocked and randomly assigned to a diet with an addition of 0.4% added fat during the last 50 d of gestation. The fat sources were rich in the monounsaturated fatty acid (MUFA) oleic acid, or the polyunsaturated fatty acids (PUFA) EPA and DHA. After lambing, ewes and lambs were placed on the same pasture. Colostrum samples were taken at lambing for fatty acid analysis. Milk yield, composition and fatty acid profile were analyzed 30 d postpartum. Data was analyzed using a mixed model (SAS 9.4) with treatment, time and their interaction as fixed variables and ewe within pen as a random variable. There was a treatment by time interaction (P < 0.05) with PUFA yielding greater EPA and DHA concentrations than MUFA at lambing, but not at 30 d in lactation. A treatment by time interaction (P < 0.05) was also observed for trans-9 18:1, trans-10 18:1, cis-15 18:1 and conjugated linoleic acid (CLA) isomers other than trans-10, cis-12 18:2, and cis-9, trans-12 18:2, with PUFA ewes having significantly greater concentrations than MUFA ewes at lambing, but not at d 30. A treatment by time interaction (P < 0.05) was also observed for cis-12 18:1, with PUFA ewes having lower concentrations than MUFA ewes in colostrum and at d 30. The concentrations of fatty acids with 6–10 carbons were significantly increased (P < 0.05) in colostrum and milk of PUFA ewes. Neither milk yield nor milk protein, fat, lactose, solids, somatic cell count, milk urea nitrogen, energy corrected milk or net energy of lactation were different (P > 0.05) among treatments. These results suggest that supplementation of EPA and DHA during late gestation alters the fatty acid profile of colostrum and milk, even 30 d after stopping the supplementation, but not milk yield or composition. Future research should investigate the effects of supplementing higher doses of EPA and DHA.