Uranium (U) is a non-essential and toxic metal for plants, which have the ability to take up uranyl ions from the soil and preferentially accumulate them in the roots. We showed that the bulk of the radionuclide accumulates in the root insoluble proteome of Arabidopsis plants challenged with U. Therefore, to elucidate new molecular mechanisms related to U stress response and tolerance, we used label-free quantitative proteomics to analyze the dynamics of the root membrane- and cell wall-enriched proteome under U stress. Of the 2,802 proteins identified, 458 showed differential accumulation in response to U. Biological processes affected by U include response to stress, amino acid metabolism, and previously unexplored functions associated with membranes and the cell wall. Indeed, our analysis supports a dynamic and complex reorganization of the cell wall in response to U stress, including lignin and suberin synthesis, pectin modifications, polysaccharide hydrolysis, and Casparian strips formation. Water flux through aquaporins and vesicular trafficking were also significantly perturbed by U stress. Finally, the abundance of metal transporters and iron, calcium, and other metal-binding proteins was affected by U. These proteins may play a role in controlling the fate and toxicity of U in plants.