Vitamin D supplementation is widely recommended for animals and humans. However, its effects on extra-skeletal disorders have not been well proven. Our research aimed to analyse the effect of supplementation with standard and increased doses of vitamin D on the health status, biochemical and haematological parameters of blood as well as on the level of expression of genes metabolising vitamin D and the Vdr gene in the kidney, liver, fat and brain of rats of different sexes. 36 Wistars rats (18 males and 18 females) divided into three supplementation groups (I-O vitamin D, II- 1000 U / kg of vitamin D, III - 5000 U / Kg of vitamin D) were analysed. Vitamin D supplementation had little effect on growth traits and biochemical blood indices; slight decrease in a heart size was observed in females supplemented with high doses of vitamin D (p = 0.0075), moreover male rats tended to have increased level of triglyceride (TG) (p = 0.0516)). In our study we observed that vitamin D supplementation did not change the expression of Vdr gene in any of analysed tissue. However, we noticed evident downregulation of Cyp27b1 gene by vitamin D supplementation in the liver and kidney in a dose-dependent manner. Additionally, we observed that in the female's liver, Vdr and Cyp2r1 were upregulated, in the female's kidney Vdr was upregulated while Cyp27b1 was downregulated compared to males. Moreover, in the female's brain and fat Cyp27b1 and Cyp2r1 tended to be upregulated compared to males. Our results confirm that Vdr and vitamin D metabolising genes are regulated by sex in the kidney and several extra-renal tissues. Copyright © 2021 Elsevier Ltd. All rights reserved.