The objective was to determine the effects of supplementation of protected conjugated linoleic acid (CLA), CLA-20 comprising 10% each of cis-9, trans-11 and trans-10, cis-12, on milk production and fatty acid profiles in plasma and milk in lactating dairy cows. Five mid-lactation, multiparous crossbred Holstein Friesian cows with average 402${\pm}$20 kg BW were used in a 5${\times}$5 Latin square design for 21-d periods. Cows were given a total mixed ration (TMR) and supplemented with CLA-20 at 0, 20, 40, 80 and 160 g/d. The results showed that dry matter intake depression occurred in cows supplemented with CLA-20 at 160 g/d. Milk production slightly increased when CLA-20 supplementation was at 20, 40 and 80 g/d. However, 3.5% fat-corrected milk (FCM) was not affected by CLA-20 supplementation. Increased levels of CLA-20 supplementation resulted in a significantly decreased percentage of milk fat. Plasma concentrations of fatty acid were not altered by the amounts of CLA-20 supplementation except for the concentration of trans-10, cis-12 CLA. For all dietary treatments, percentages of fatty acids (C4:0, C6:0, C8:0, C13:0, C14:0 C14:1 C15:0 C15:1 C16:0, C16:1, C18:1n9t, C18:2n6t, C18:2n6c, C20:0, C18:3n6, C18:3n3, C20:1 and C20:3n6) in milk fat were similar. Concentrations of C10:0, C11:0, C12:0 and C18:1n9c were decreased cubically and C18:0 was elevated linearly (p<0.01) according to the increased amounts of CLA-20 supplemented. The linear increase was observed for cis-9, trans-11 CLA (0.62, 1.17, 1.94, 1.87 and 1.82% of total fatty acid), trans-10, cis-12 CLA (0.01, 0.63, 0.67, 0.93 and 0.95% of total fatty acid) and total CLA (0.80, 2.25, 3.16, 3.97 and 3.94% of total fatty acid) in milk fat from 0 to 160 g/d of CLA-20 supplement. In conclusion, concentration of cis-9, trans-11 CLA in milk fat was concomitantly elevated at an increasing rate with the increased amounts of CLA-20. Based on the results in this study, supplementation of CLA-20 at 80 g/d optimally enhanced total CLA in milk fat.