Feeding unsaturated fatty acids (FA) as Ca salts has been proposed as a way to protect them against ruminal biohydrogenation. However, dissociation of Ca salts in the rumen limits the efficiency of this protection. Industrial processes used to obtain Ca salts of FA lead to the production of commercial feeds with a range of different particle sizes. We hypothesized that unsaturated FA in large particles are physically protected against ruminal biohydrogenation. Calcium salts of polyunsaturated FA were obtained from Virtus Nutrition LLC (Corcoran, CA). On a FA basis, the preparation contained 22.4% 18:1n-9, 14.7% 18:2n-6, 31.9% 18:3n-3. The product was sieved through a 1.9-mm screen. The retained particles were saved, and identified as coarse Ca salts. The particles less than 1.9-mm were ground through a 0.864-mm sieve, and identified as fine Ca salts. A mixture of unprotected FA, as triglycerides, with a composition similar to that of the Ca-salts served as control. Eight Holstein cows were used in a 4 × 4 Latin square design. Treatments were N-CTL) ruminal dosing of unprotected FA, used as negative control; FCS) ruminal dosing of fine Ca salts; CCS) ruminal dosing of coarse Ca salts; and P-CTL) abomasal dosing of unprotected FA, used as positive control. Treatments were adjusted to provide 600 g FA per day, and were offered in 2 equal boluses at 1000 and 1600h for 14 d, followed by 14-d washout intervals. Pre-planned contrasts were used to compare CCS with i) N-CTL; ii) FCS; and iii) P-CTL. Milk fat content of 18:2n-6 was 25.4 mg/g for CCS, and was lower in cows fed N-CTL (16.5 mg/g; P = 0.01) or FCS (15.0 mg/g; P < 0.01); and higher in cows fed P-CTL (35.2 mg/g; P < 0.01). The concentrations of 18:3n-3 in milk fat was 28.9 mg/g for CCS, and was lower in cows fed N-CTL (7.0 mg/g; P = 0.01) or FCS (14.6 mg/g; P = 0.08, tendency); and higher in cows fed P-CTL (92.2 mg/g; P < 0.01). In conclusion, feeding CCS appeared to have partially prevented ruminal biohydrogenation, and increased milk fat content of polyunsaturated FA as compared with dietary unprotected oil or FCS.