The aim of the experiment was to determine the AME(N) value of feed grade glycerol for laying hens on the one hand and on the other hand to assess the effect of glycerol on egg production, egg weight, egg mass, egg yolk and egg-white ratio, nutrient content of egg (energy, protein and fat content), and fatty acid profile of yolk. The experiment was conducted with 48 Tetra SL laying hens, placed into individual metabolic cages. Birds were divided into four groups and they consumed a mash laying hen diet supplemented with 0 (control), 5.0, 7.5 or 10% glycerol. The AME(N) content of feed grade glycerol (glycerol content:86.8%) was determined as 15.36 MJ/kg, which is 95.8% of gross energy (15.59 MJ/kg). Feeding the diet supplemented with 5.0, 7.5 or 10% glycerol did not significantly influence egg production, egg weight and egg mass. Furthermore, it did not affect significantly egg yolk and egg-white proportion, energy, protein and fat content of eggs. But, supplementation of glycerol influenced significantly contents of several fatty acids in egg yolk. Supplementation of 5.0 and 7.5% glycerol increased significantly myristic (C14:0) and arachidic acid (C20:0) content. In contrary, content of heptadecanoic acid (C17:0) was decreased significantly for supplementation of 5% glycerol. In summary, supplementation of glycerol increased the proportion of saturated fatty acids, which is negative for human nutrition. Supplementation of glycerol significantly decreased the content of heptadecanoic acid (C17:1) and increased the content of arachidonic acid (C20:4). Supplementation of 7.5% glycerol significantly increased the contents of linolenic acid (C18:3) and eicosatrienoic acid (C20:3). Simultaneously, feeding the diet with 5% glycerol significantly reduced the contents of docosatetraenoic acid (C22:4) and docosahexaenoic acid (C22:6). This resulted in a narrower (relatively 11%) n-6/ n-3 ratio, which is a favorable effect in view of the nutritive value of the egg. Although glycerol supplementation caused significant changes in the case of some fatty acids, the rate of changes was not remarkable.