The aim of the study was to evaluate the potential effect of different levels of sumac (Rhus coriaria L.) seed powder and ginger (Zingiber officinale) root powder on egg yolk fatty acid composition, blood/yolk cholesterol in laying hen. A total of 63 (ATAK-S: Domestic Turkish Laying Hens) laying hens (average weight: 1470 g each hen, 25-weeks of age) were assigned to seven treatment diets including sumac seed (S) and ginger root powder (G) at 0 g/kg (control), 10 g/kg (S1), 20 g/kg (S2), and 30 g/kg (S3); 10 g/kg (G1), 20 g/kg (G2), or 30 g/kg in rations respectively, for 8weeks. After a two-week adaptation period to cages, the hens were allocated to 7 groups with 9 replicates of 1 hen in per cage each. The replications were allotted equally into the upper and lower cages to minimize the effects of cage level. In this study, egg yolk cholesterol had a decrease (p <0.05) in supplemented diet (sumac seed and ginger root powder). Fatty acid content in yolk; saturated fatty acid, monounsaturated fatty acids, polyunsaturated fatty acids and rate of n6/n3 were not significant (p <0.05). However, dietary supplementation with sumac and ginger powder reduced and yolk/blood cholesterol concentrations in laying hens. Supplementation of sumac and ginger affected on HDL, there was found a significant effect (p<0.05) in treatment groups. Moreover, LDL positively decreased in all treatment groups compared with the control group. The findings of this study suggested that feeding sumac and ginger tend to be decreasing cholesterol levels in both yolk and blood on laying hens. It can be concluded that ginger root and sumac seed powder can be used as an effective feed additive to improve fatty acid composition and yolk and blood cholesterol in ATAK-S laying hens.