The present study was conducted to investigate the effect of dietary supplementation of Echinacea purpurea on performance, yolk oxidative stability, ileal microflora and immune responses of laying hens. A total of 150 Hy-line W-36 Leghorn hens of 44-wk of age were randomly distributed among 5 replicate pens of 5 experimental treatments. Dietary treatments consisted of the levels of 0, 0.25, 0.50, 0.75 or 1% of Echinacea purpurea powder that fed during a 77 d feeding trial including 7 days for adaptation period and 70 days as main recording period. Hen-day egg production and egg weights were recorded daily, but all performance parameters were reported as 35 days intervals. At d 70 of main recording period, two randomly selected hens per replicate were slaughtered for carcass analysis and ileal microbial count. There were significant (P 0.05) differences between treatments in the light of egg production during both 35 d periods and egg mass only during the second 35 d period. Echinacea supplementation caused significant (P 0.05) increase in egg production and egg mass during both 35 d periods. In contrast, dietary addition of Echinacea had no considerable effect on egg weight, feed consumption and feed conversion ratio, but tended (P = 0.07) to improve feed conversion efficiency. Among the egg quality parameters, only yolk color index was affected (P 0.05) by dietary treatments during the first 35 d period. Also, experimental diets tended to affect yolk index during the first (P = 0.17) and second (P = 0.08) 35 d periods so that, Echinacea diets caused numerical deceases in yolk index. However, other egg quality measurements were not influenced by dietary treatments. Dietary inclusion of Echinacea decreased (P 0.05) serum triglycerides during the first 5 wk, and serum cholesterol was also decreased (P 0.01) at d 35 by Echinacea supplementation. There was significant difference between dietary treatments in the light of serum cholesterol concentration at d 70 of trial. Interestingly, serum HDL was increased in both 35 d as the result of dietary Echinacea supplementation. Furthermore, egg yolk cholesterol was affected (P 0.05) by dietary treatments and Echinacea powder increased (P 0.05) yolk triglycerides compared with control group. On the other hand, egg yolk cholesterol wasn’t affected in Echinacea-supplemented group compared with control hens but there was significant difference between treatments. Although lymphocyte, heterophil and eosinophil proportions in peripheral blood weren't affected by Echinacea supplementation, however, the levels of 0.50 and 0.75% of Echinacea increased (P 0.05) blood monocytes. Dietary supplementation of Echinacea powder modified ileal microbial ecosystem so that decreased (P 0.05) total microbial count and E.coli in ileal contents. Also, Echinacea powder caused a significant (P 0.01) increase in liver weight as a percentage