The current study was conducted to evaluate the effect of different levels of fat and milk thistle ( Silybum marianum ) meal on performance, egg quality, blood biochemical parameters, ileal microbial enumeration, intestinal histology, antioxidant indices of blood and liver, and fatty liver indices of laying hens. A total of 300 laying hens were randomaly distributed among 12 experimental treatments with 5 replicotes of 5 birds each. Dietary treatments consisted of four fat sources (fish oil, sunflower oil, poultry oil and fat powder) and three levels of milk thistle meal (0, 1.5 and 3%) that fed during a 80 d feeding trial including 10 days as adaptation period and 70 days as main recording period. Performance parameters including feed intake, feed conversion ratio, egg production percentage, egg weight, egg mass and also egg quality were calculated on 35 th day and 70 th day of experiment.To investigate immunological responses, birds were injected whit sheep red blood cells (SRBC) twice, at an interval of ten days. After that, hens were exsanguinated respectively on the 6 th day after the first injection and on the 12 th day after the second injection. Moreover, Newcastle disease virus was injected 10 days before the first injection and then sera were taken on 6 th day after injection. In addition, differential leukocyte counts were evaluate at the end of experiment. Additionally on 70 day of experiment, two birds of each cage were sacrificed to evaluate the relative weight of internal organs, enumeration of intestinal microbial counts as well as hepatic and jejunal morphology. The results showed that fish and sunflower oils led to decrease plasma cholesterol and liver glycogen contents, and increase the relative weight of spleen (P 0.05). Fish oil and poultry oil led to decrease the enzymatic activity of SGOT, relative weight of liver and hepatic fat percentage (P 0.01). Fish oil led to increase antibody titer against SRBC and Newcastle disease virus in the second response, as well as the percentage of egg production in the first and entire enperimental periods. It also decreased liver tissue integrity (P 0.01) and egg cholesterol (P 0.05) and also improved the color of egg yolk (P 0.05). Fat powder increased ALP enzyme activity, feed conversion ratio in the entire period and palmitic acid in the liver (P 0.05). Fish oil increased the percentage of eicosapentaenoic acid and docosahegsaenoic acid, sunflower oil let to increase linoleic acid, and poultry oil led to increase oleic acid in the liver (P 0.01). Feeding at least 1.5 % of milk thistle meal increased the antibody titer against SRBC and eicosapentaenoic acid (P 0.05). In addition, using 3 % of milk thistle meal improved the serum biochemical parameters (P 0.01), percentage of lymphocytes (P 0.05), performance of laying hens (P 0.01), and also decreased the relative weight of liver (P 0.01) and ileal Escherichia coli count. Furthermore, feeding the highest level of milk thistle meal improved the ratio of villus height to crypts depth (P 0.01), Kupffer cell numbers (P 0.0001), eggshell hardness and thickness at the second period of the experiment (P 0.05), and also decreased egg yolk cholesterol (P 0.01), and palmitic acid in the liver (P 0.01). The lowest relative weight of liver and ileal Escherichia coli count (P 0.01) were observed in hens fed on the highest level of milk thistle meal. However feeding fish oil, poultry oil and sunflower oil, specially when supplemented whit 3% milk thistle meal, caused higher productive performance, the best immune reonses were observed in birds fed diets supplemented whit fish oil and 3% milk thistle meal. In addition, dietary adminasteration of fish oil and poultry oil as well as the highest level of milk thistle meal could improve the fatty liver indices, markedly. Keywords: Fat sources, Milk thistle meal, Pefprmans, Intestinal histology, Fatty liver Laying hens