This study was designed to investigate the effects of the usage of glycerol from biodiesel production from soybean oil in laying hen diets on laying performance, egg traits, heterophils to lymphocytes ratio (H/L), some blood parameters and antibody production to SRBC. A total of 180 Lohmann Brown laying hens 39 weeks of age were allocated to four dietary treatments with one control group and three treatment groups and fed for 16 weeks. Each group was divided into five replicates as subgroups, comprising of 9 hens each. Glycerol was used at the level of 2.5, 5 and 7.5% in the diets of the first, second and third treatment groups, respectively. The diets were formulated to be isocaloric and isonitrogenous. Dietary treatments did not significantly affect body weight, egg production, egg weight, feed efficiency, mortality, egg albumen index, egg yolk index and egg Haugh unit, yolk weight percentage, exterior egg quality characteristics, excreta moisture, H/L ratio, blood parameters and antibody production to SRBC. Hens fed diets with 7.5% glycerol consumed significantly less feed than those of the other groups. Egg yolk cholesterol concentration was significantly higher for hens fed diets with 5 and 7.5% glycerol as compared to those of the other groups (P < 0.01). The ratio of monounsaturated fatty acids (MUFA) to saturated fatty acids (SFA) in eggs was decreased (P < 0.01) with dietary glycerol supplementation. The inclusion of glycerol had no significant effects on blood parameters, H/L ratio, antibody titers to SRBC and excreta moisture. It is concluded that glycerol can be used at 2.5% in the diets of laying hens without adverse effects on the measured parameters. Dietary glycerol at the levels of 5 and 7.5% increased egg yolk cholesterol and decreased the ratio of MUFA/SFA without affecting performance, other egg traits, immune response, H/L ratio, blood parameters and excreta moisture. © 2010 Elsevier B.V. All rights reserved.