Thirty-one Holstein cows (six ruminally cannulated) were used to evaluate milk fatty acids (FA) composition and conjugated linoleic acid (CLA) content on three dietary treatments: 1) total mixed rations (TMR), 2) pasture (Avena sativa L.) plus 6.7 kg DM/d of corn-based concentrate (PCorn), and 3) pasture plus PCorn with 0.8 kg DM/d of Ca salts of unsaturated FA replacing 1.9 kg DM/d of corn (PFat). No differences were found in total (22.4 kg/d) or pasture (18.5 kg/d) dry matter intake, ruminal pH, or total volatile fatty acids concentrations. Fat supplementation did not affect pasture neutral detergent fiber digestion. Milk production did not differ among treatments (19.9 kg/d) but 4% fat-corrected milk was lower for cows fed the PFat compared to cows fed the TMR (16.1 vs. 19.5 kg/d) primarily because of the lower milk fat percentage (2.56 vs. 3.91%). Milk protein concentration was higher for cows fed the TMR than those on both pasture treatments (3.70 vs. 3.45%). Milk from the cows fed the PCorn had a lower content of short- (11.9 vs. 10.4 g/100 g) and medium-chain (56.5 vs. 47.6 g/100 g) FA, and a higher C18:3 percentage (0.07 vs. 0.57 g/100 g) compared with TMR-fed. Cows fed the PFat had the lowest content of short- (8.85 g/100 g) and medium-chain (41.0 g/100 g) FA, and the highest of long-chain FA (51.4 g/100 g). The CLA content was higher for cows in PCorn treatment (1.12 g/100 g FA) compared with cows fed the TMR (0.41 g/100 g FA), whereas the cows fed the PFat had the highest content (1.91 g/100 g FA). Pasture-based diets increased the concentrations of long-chain unsaturated FA and CLA in milk fat. The partial replacement of corn grain by Ca salts of unsaturated FA in grazing cows accentuated these changes. However, those changes in milk FA composition were related to a depression in milk fat.