This study was performed to examine an efficient way to use oil by-product which is obtained during the purification process of conjugated linoleic acid (CLA) from safflower oil. The CLA by-product (CBP) was fed to the laying hens to accumulate CLA in the egg yolk. Egg yolk samples from 3 different dietary groups were analyzed: control; CBP, 2%; and CLA-80 (which contained 80% free form of CLA), 2%. Fatty acids from the yolk were analyzed by gas chromatography, and the parameters of egg quality were determined. During the feeding trial, there was little alteration in the egg quality and egg production of the tested groups. The CLA contents in the test group with CBP, which contain CLA as glyceride form, significantly increased in the first week of feeding and had the highest level among the tested groups throughout the feeding trials, whereas the CLA group showed an increase in CLA contents from the third week. Moreover, the contents of total CLA isomers in CLA-80 and CBP oils were decreased by 12.92 and 0.51% after heat treatment in 48 h, and the contents of linoleic acid (LA) isomer in LA-80 (which contained 80% free form of LA) and LA by-product (LBP) oils were decreased by 19.63 and 5.78%, respectively. It was confirmed that a major form of fats in CBP was mainly esterified forms, whereas the fats in CLA-80 and LA-80 were composed of free fatty acids. There was no significant difference in the egg quality and production among the tested groups. It is meaningful that the oil by-product could be utilized as a source for functional foods of animal origin without influencing egg quality and production.