The role of calcium (Ca) on the cellular distribution of U(VI) in Brassica juncea roots and root-toshoot translocation was investigated using hydroponic experiments, microscopy, and spectroscopy. Uranium accumulated mainly in the roots (727−9376 mg kg−1) after 30 days of exposure to 80 μM dissolved U in water containing 1 mM HCO3 − at different Ca concentrations (0−6 mM) at pH 7.5. However, the concentration of U in the shoots increased 22 times in experiments with 6 mM Ca compared to 0 mM Ca. In the Ca control experiment, transmission electron microscopy−energy-dispersive spectroscopy analyses detected U−P-bearing precipitates in the cortical apoplast of parenchyma cells. In experiments with 0.3 mM Ca, U−P-bearing precipitates were detected in the cortical apoplast and the bordered pits of xylem cells. In experiments with 6 mM Ca, U−P-bearing precipitates aggregated in the xylem with no apoplastic precipitation. These results indicate that Ca in carbonate water inhibits the transport and precipitation of U in the root cortical apoplast and facilitates the symplastic transport and translocation toward shoots. These findings reveal the considerable role of Ca in the presence of carbonate in facilitating the transport of U in plants and present new insights for future assessment and phytoremediation strategies.