Abstract. The objective of this experiment was to replace soybean oil with glycerol in feeding mixtures for layers and assess its effect on performance, selected parameters of egg quality and health. Layers were divided into control (C) and experimental (E1, E2) groups of 24 layers each. The experiment was performed from week 20 until week 44 of layers’ age. Soybean oil replacement with glycerol reached 50 and 100 % in experimental feeding mixtures E1 and E2, as compared to the control. Oil replacement with glycerol in experimental groups resulted in a significant decrease (P≤0.01) in weight of eggs, as compared to the control group – C (65.5±5.780 g), E1 (64.53±4.640 g) and E2 (63.66±5.610 g), and egg shell stability (N/cm²) – C 33.79±6.557, E1 31.96±6.995 and E2 31.8±6.621. The difference in the weight of egg shell between groups C (7.13±0.687 g) and E2 (6.85±0.670 g) was highly significant (P≤0.01), the difference between E1 (7.00±0.820 g) and E2 groups for the same parameter was significant (P≤0.05). No significant differences were found between mean values of the weight of egg yolk and white. With regard to feed consumption, 50 % replacement of oil with glycerol did not increase significantly the daily consumption of a feeding mixture per layer (1 egg) – C 122 g (132 g) and E1 123g (136 g), as compared to 100 % replacement in E2 131 g (150 g). The analysis of blood plasma after the prolonged continuous administration of glycerol in a feed did not show any negative effect on layers’ health.