Iodine is one of the trace elements which are essential for mammalian life. The major objective of iodine biofortification of plants is to obtain food rich in this trace element, which may increase its consumption by various populations. Additionally, it may reduce the risk of iodine deficiency diseases. In this research for the first time we have assessed the bioavailability of iodine from raw or cooked carrot biofortified with this trace element on iodine concentration in selected tissues and various biochemical parameters as well as mRNA expression of some genes involved in iodine metabolism in Wistar rats. Statistically, a significantly higher iodine level was determined in urine, faeces and selected tissues of rats fed a diet containing biofortified raw carrot as compared to a diet without iodine and a diet containing control cooked carrot. Biofortified raw carrot significantly increased triiodothyronine concentration as compared to animals from other experimental groups. The highest thyroid stimulating hormone level was determined in rats fed control cooked carrots. mRNA expression of selected genes was affected by different dietary treatment in rats' hearts. Biofortified raw and cooked carrot could be taken into account as a potential source of iodine in daily diets to prevent iodine deficiency in various populations.