Sunflower (Helianthus annuus) can potentially be used for uranium (U) phytoremediation. However, the factors influencing the absorption of U and its subsequent distribution within plant tissues remain unclear, including the effect of silicon (Si) which is known to increase metal tolerance. Here, using hydroponics, the effect of Si on the distribution and speciation of U in sunflower was examined using synchrotron-based X–ray fluorescence and fluorescence-X-ray absorption near-edge spectroscopy. It was found that ~88% of U accumulates within the root regardless of treatments. Without the addition of Si, most of the U appeared to bind to epidermis within the roots, whereas in the leaves, U primarily accumulated in the veins. The addition of Si alleviated U phytotoxicity and decreased U concentration in sunflower by an average of 60%. In the roots, Si enhanced U distribution in cell walls and impeded its entry into cells, likely due to increased callose deposition. In the leaves, Si induced the sequestration of U in trichomes. However, Si did not alter U speciation and U remained in the hexavalent form. These results provide information on U accumulation and distribution within sunflower, and suggest that Si could enhance plant growth under high U stress. Environmental ImplicationAccumulation of U in the aerial parts of plants is crucial for U phytoremediation. However, consistent with the previous studies, we have found that the roots are the main organ for U accumulation in sunflower, with the present study identifying the possible reasons for this limited root-to-shoot translocation of U in sunflower. More importantly, our results indicated the addition of Si substantially alleviates U toxicity by reducing U concentration and altering its distribution in sunflower. These findings offer novel insights for improving plant growth under high U stress.