The effects of dietary inclusion of distillers dried grains with solubles in laying hen diets with and without exogenous enzyme mixture (EEM) on performance and egg characteristics were evaluated. One of the main objectives of this study was to examine the effects of distillers dried grains with solubles (DDGS) and enzyme cocktail on egg yolk fatty acids. The study used total of 144 Hisex Brown laying hens in a 4 x 2 factorial arrangement, including four levels of DDGS (0, 6, 12, and 18% of diet) and two levels of enzyme cocktail (0 or 250 mg EEM/kg of diet) through 32-42 weeks of the age. The inclusion of 18% DDGS was associated with the worst (p 0.001) egg production and the lowest daily feed intake. Numerically, hens fed 6% DDGS diet consumed more feed and had the greatest egg production. The best feed conversion ratio (FCR) was recorded in the control, while the worst was recorded in the 18% DDGS group. Compared with EEM-free diets, EEM supplementation improved FCR by about 2.79%, but the difference was not significant. Shell thickness and shell percentage were significantly increased in hens fed 6% DDGS diet compared to other groups. Egg weights in the 6% and 12% DDGS groups were significantly higher than those in the control and 18% DDGS groups. Non-significant increases in shell and albumin percentages were recorded in groups fed EEM-supplemented diets. The interaction effect of DDGS and EEM was significant (p 0.01) for the majority of egg characteristics. As dietary DDGS level increased, yolk color density increased. Elevating DDGS level up to 18% increased yolk cholesterol, total fat, and total unsaturated fatty acids. The effects of EEM supplementation on egg yolk fatty acid composition and the interaction effects between DDGS and EEM were not significant. Considering these results, it could be concluded that DDGS is an acceptable feed ingredient in layer diets and that the maximum inclusion level of DDGS should not exceed 12% of the whole diet.