The present study was conducted to investigate the effect of dietary choline and carnitine as the lipotropic agents, on performance, immune responses, serum and biochemical parameters and antioxidant capacity of liver and yolk in laying hens fed diets with incremental levels of metabolizable energy (ME). A total of 240 Hy-line W-36 laying hens of 46 weeks of age were used during a 77 days trial period. The birds were randomly distributed among 5 replicates (5 hens per each cage replicate) of each of 9 dietary treatments. Experimental treatments were consisted of 3 different ME levels (control value, and 100 and 200 kcal/kg greater) and 3 situations of lipotropic factors (control diets, and diets supplemented choline or carnitine) based on a 3× 3 factorial arrangement of treatments. Performance parameters including feed intake, production percentage, egg weight, egg mass, feed conversion ratio (FCR)and egg quality indices were recorded periodically (two 35d intervals). At the final trial day, two birds from each cage were slaughtered to measure relative weights of liver, gallbladder, pancreas, heart and spleen. Antibody titers against Newcastle disease virus (NDV) and sheep red blood cell (SRBC) were measured after respective inoculations. In addition, liver and yolk samples were analyzed for lipid metabolites and malondialdehyde (MDA) concentrations. Results showed that dietary supplementation with both lipotropic factors caused significant (P 0.01) decrease in feed intake with the least feed consumption assigned to the birds on choline-supplemented diets.Supplementation of choline and carnitine in the first 35 d (P 0.01), second 35 d and entire exprimental (P 0.05) periodsimproved FCR values. Egg production and egg weight were not affected by dietary ME levels, however, supplementation of choline into the diets tended (P=0.0554) to improve egg production. Among the egg quality indices, dietary supplementation of lipotropic factors reduced (P 0.05) haugh unit during first 35 d and dietary supplementation of carnitine resulted in a marked deacrease in haugh unit in the highest ME diets, coused signifying (P 0.05) lipotropic factors × ME levels intraction. Eggshell breaking strength and thickness, yolk color and shape index were not affected by dietary treatments.Increase in dietary ME level inceased (P 0.05) relative spleen weight, while supplemental choline decreased liver weight. Although increasing dietary ME level increased (P 0.01) liver fat, yolk triglycerides, and liver and yolk MDA concentrations, dietary supplementation of lipotropic factors significantly (P 0.01) reduced the mentioned parameters. serum concentrations of cholestrol and LDL increased (P 0.01) by dietary ME levels, however, serum triglycerides reduced (P 0.001) as the result of dietary supplementation with carnitine. Antibody response to SRBC andNDV were not affected by ME levels, however, supplementation of lipotropic factors into the diets improved (P 0.05) NDV antibody titer at 14 th d after vaccination. The present findings indicated that supplementation of high-ME diets with lipotropic agents improves feed efficiency and has a beneficial impact on serum lipid parameters. In addition, supplemental diets choline and carnitine could improve antioxidant capacity of egg yolk and liverhealth in laying hens. Keywords: Laying hens, Choline, L-carnitine, Dietary ME level, Performance, Immune responses, Egg quality indices