Uranium (U) is a naturally occurring radionuclide that is toxic for plants. The aim of this study was to gain insights into the physiological and cellular responses of roots to U stress. We analyzed the effects of uranyl nitrate on the architecture and physiology of Arabidopsis thaliana roots using different staining procedures and reporter genes. Also, we examined the homeostasis of inorganic phosphate (Pi) and Fe during U stress. We showed that, at a sub-toxic dose, U stimulated the apex mitotic activity, resulting in improved primary root growth and reduced secondary root formation. At a toxic level, U arrested primary root growth and increased the formation of secondary and higher-order lateral roots. U stress was linked with a depletion of Pi and a redistribution of Fe in root tissues, together with the production of ROS and nitric oxide. Also, U triggered the deposition of the defense polymers callose and lignin. These results showed that part of the radionuclide effects are linked with the signaling cascade of Pi sensing in the root apex. Other mechanisms involved in U toxicity are likely related to perturbations of Fe homeostasis and direct deleterious effects of U on root components.