Uranium and Cesium 137 (137Cs) are both radionuclides found in the environment as a result of their accidental dispersion and/or natural presence. Consequently, some human populations are exposed to these radioelements mainly through chronic ingestion. Chemical and/or radiological toxicity of uranium and 137Cs has been reported in kidney, liver and brain that play key-roles in vitamin D metabolism. Beside, alterations of both bone and phospho-calcium homeostasis have been reported after an acute or chronic contamination with uranium or 137Cs. However, vitamin D, the major regulator of mineral homeostasis has never been studied up to now. The aim of this work was to investigate in vivo the effects of depleted (DU) or enriched uranium (EU) and of 137Cs on vitamin D3 biosynthetic pathway in liver, kidney and brain. An experimental animal model was used for the first time to demonstrate that chronic exposure with environmental doses of 137Cs and uranium could decrease the vitamin D active form level (1,25(OH)2D3) and lead to molecular modifications of cytochromes P450 (CYPs) enzymes involved in this metabolism and associated nuclear receptors. We demonstrated that both UA and UE contamination affected VDR (vitamin D receptor) and RXRα (retinoid X receptor alpha) expression, and consequently could modulate the expression of vitamin D target genes involved in calcium homeostasis in kidney. These results suggest that these effects could be due to the chemical toxicity of uranium. On the contrary, the main molecular targets of 137Cs are CYPs involved in Vitamin D3 biosynthesis (CYP2R1, CYP27B1) in liver and brain. In this adult rat model such perturbations were not associated with a dysregulation of mineral homeostasis. Conversely, chronic exposure with 137Cs during postnatal development induce alterations of vitamin D metabolism associated with modifications of bone and phospho-calcium homeostasis, suggesting a greater susceptibility of the growing organism to 137Cs contamination compared to adult.