Background and Aims <p>Silicon (Si) can mitigate aluminum (Al) toxicity in rice (<i>Oryza sativa</i> L.), but the underlying mechanisms still remain unclear, particularly under prolonged Al stress. This study aims to elucidate the interaction between Si and Al in rice and to explore the role of Si accumulation in enhancing resistance to Al toxicity.</p> Methods <p>Three rice genotypes were used: a low Si-accumulating mutant (<i>lsi2</i>), its wild type (cv. Taichung-65), and a conventional cultivar (cv. Wuyunjing 30). We investigated the effects of endogenous Si on the acquisition, translocation, and distribution of Al in rice through 22-day experiments involving both Si-Al coexistence and Si-Al separation.</p> Results <p>Si accumulation in roots decreased Al acquisition and root Al concentration by inhibiting Al radial transport in roots and reducing the Al-binding capacity of root cell walls. The consequent reduction in shoot Al concentration was primarily due to this decrease in root Al acquisition rather than a lower root-to-shoot Al translocation factor. Meanwhile, Al stress inhibited Si acquisition by roots and reduced shoot Si concentration, but significantly enhanced root Si concentration which increased with rising Al concentration in the culture medium. Furthermore, a positive Si-Al interaction was observed, substantially improving rice growth, with OsLsi2 playing a critical role in this process.</p> Conclusions <p>Under prolonged Al stress, the accumulation of Si in roots is a key to reduce Al acquisition by roots, decrease Al concentration in rice, and promote plant growth.</p>