Abstract: Objective of this study was to determine the comparative effectiveness of linoleic and linolenic acid in enhancing CLA in milk when fed at the same levels to mid lactation dairy cows. Forty dairy cows were randomly assigned to 4 treatments. Cows in four treatments were fed a basal diet that was supplemented oils differed in their linoleic and linolenic acid contents. The four treatments were Low linoleic and linolenic acid(LLALEA), high linoleic(HLA), High linolenic acid(HLEA) or High linoleic and linolenic acid(HLALEA). Experimental duration was 9 weeks and measurements were made during the last 6 weeks. Dry matter intake, energy corrected milk (ECM) , milk fat and protein contents were not differ among treatments. The proportions of C18∶1 trans-11 (TVA) and C18∶2 cis-9, trans-11 (CLA) were 1.48, 6.19, 3.04, and 4.53 (P=0.01), 0.64, 2.39, 1.60, and 1.81% of total fatty acid methyl esters in LLALEA, HLA, HLEA, and HLALEA, respectively. The proportions of unsaturated fatty acids were 27.9, 33.6, 36.4, and 33.8 (P=0.001) in LLALEA, HLA, HLEA, and HLALEA, respectively. Feeding free oil rich in linoleic acid is 100 and 50% more effective in enhancing TVA and CLA in milk fat, respectively, than feeding free oil rich in linolenic acid fed at the similar levels in the diet.The milk fat content of CLA c-9,t11 across the whole treatment increased linearly with increased intake of dietary LA. Milk fat concentration of TVA in HLEA,HLALEA and HLA were 1.56,3.05 and 4.71 g/100 g higher than LLALEA respectively ,while CLA c-9,t11 were 2.5,2.8 and 3.7 times respectively. Therefore, dietary LA ,which was used as the main substrate for CLA endogenous synthesis via SCD in the mammary gland, is more effective than LEA in enhancing milk CLA c-9,t11 concentration.