The objective of this experiment was to replace soya bean oil with glycerol in laying hen diets and assess the change's effect on performance, parameters of egg quality and the egg fatty acid profile. A total of 60 44-week-old Hy-Line W36 laying hens were distributed according to a completely randomised experimental design into four treatments consisting of glycerol substitutions for soya bean oil dietary at varying inclusion levels (0%, 25%, 50% and 75%), with five replicates of three birds each. Dietary treatments had no significant effect on BW change, egg production, feed intake, feed conversion ratio, egg weight and egg mass of laying hens. The inclusion of glycerol in the diet of laying hens had no significant effect on egg specific gravity, eggshell breaking strength, eggshell weight, eggshell thickness, egg shape index, albumen index, yolk index, haugh unit, albumen pH, yolk pH and egg yolk colour values. The inclusion of glycerol in the diet of laying hens had no significant effect on palmitic, palmitoleic, stearic, oleic and linolenic acid contents of the egg yolk. The linoleic acid and polyunsaturated fatty acid contents of the egg yolk significantly decreased with the higher levels of dietary glycerol supplementation (P<0.05). The results of this study show that it is possible to replace 75% of soya bean oil (4.5% in diet) with glycerol.