We determined the effects of feeding canola oil or infusing it into the abomasum on rumen fermentation, nutrient digestibility, duodenal flows of fatty acids, and milk composition in Holstein cows. Five ruminally and duodenally cannulated Holstein cows in late lactation were used in a 3×5 incomplete Latin square design. Treatments were 1) Control: basal diet (CON), 2) Control+supplementation of canola oil at 1 kg/d in the feed (FED), and 3) Control+abomasal infusion of canola oil at 1 kg/d (INF). Compared with CON, feed intake, ruminal fermentation characteristics, ruminal and total tract digestibilities of nutrients were not significantly affected by FED treatment but duodenal flows and milk concentrations of fatty acids (FA) such as trans-11 18[ratio ]1 and cis-9 trans-11 18[ratio ]2 (conjugated linoleic acid, CLA) were increased. In contrast to the effects of FED, INF reduced feed intake, total VFA production, intestinal flows of nutrients, FA digestibility and yields of milk and milk fat. Both FED and INF significantly reduced the proportions of saturated and medium-chain FA, and increased cis 18[ratio ]1 in milk. Concentrations of 18[ratio ]2n-6 and 18[ratio ]3n-3 in milk were increased nearly 2-fold with INF relative to CON. Dietary or postruminal supplementation of canola oil to late-lactation cows reduced saturated FA and increased unsaturated C18 in milk but nutrient digestion was adversely affected with abomasal infusion of canola oil.