In resent year there has been research on benefits of conjugated linoleic acid (CLA) and docosahexaenoic acid (DHA) for human health. The effect of a fat supplement made from seaweeds meal plus palm olein meal (FS) and replacement of concentrate on milk production, and milk fatty acid (FA) profiles of dairy cows fed grass silage based diets was evaluated. Ten Holstein lactating cows were divided into a two groups. The cows in a group were fed concentrate at 40% on TDN basis (control). The cows in another group (Treatment) were supplemented 0.5㎏/day/cow of FS as a substitute for 2㎏/day/cow of concentrate. The medium chain fatty acids and DHA content of FS were 345 ㎎/g DM and 35 ㎎/g DM, respectively. Total DMI, DM digestibility and milk production as well as milk composition did not differ between the two groups. The intake of ether extract was higher (P＜0.01) in Treatment than control group (1.1 ㎏/day/cow vs. 1.3 ㎏/day/cow). The short and medium chain of FA in the milk did not differ between two groups. The trans11C18:1vaccenic acid, cis9trans11CLA and C18:3 concentrations in the milk were higher (P＜0.01) in Treatment than control group(1.88 g/100gFA vs. 1.03 g/100gFA, 0.93 g/100gFA vs. 0.51 g/100gFA and 0.48 g/100gFA vs. 0.48 g/100gFA). However, the C18:2 concentration in the milk was lower (P＜0.05) in Treatment than Control group (1.38 g/100gFA vs.1.17 g/100gFA). There were detected 0.13 g/100gFA of DHA in the milk in Treatment group. Results indicated that addition of the FS has the potential of increasing CLA and DHA in cow's milk.