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Abstract Objective Sakura extract is a natural flavonoid compound that may have potential anti-tumor effects. The paper focuses on investigating Sakuranin mechanism on bladder cancer (BC) cells. Methods BC cells (T24) were treated with different concentrations of Sakuranin, with 48-h IC50 determined. T24 cells were treated with Sakuranin at IC50, followed by assessment of cell proliferative/apoptotic/migrative/invasive activities by CCK-8, EdU and plate clone formation assays/flow cytometry/Transwell/scratch test. MMP-2 (migration and invasion-related protein) protein level was assessed by Western blot. Cell autophagy was evaluated by measuring the protein levels of autophagy markers (LC3-I/LC3-II/p62) through Western blot. The autophagy inhibitor 3-MA was used to validate the role of autophagy in the regulatory mechanism of Sakuranin in T24 cell behaviors. Furthermore, the activation of the p53/mTOR pathway in cells was detected and a combination of Sakuranin and p53 inhibitor Pifithrin-µ was adopted to explore the involvement of this pathway. Results Sakuranin decreased T24 cell proliferation/EdU positive cell percentage/colony formation number and area/migration/invasion/scratch healing/MMP-2 protein level, and accelerated apoptosis. Sakuranin elevated the LC3-II/I ratio and lowered p62 level in T24 cells. 3-MA partially averted Sakuranin-mediated repression on cell malignant behaviors. Sakuranin upregulated p-p53 and p53 levels, and decreased the p-mTOR/mTOR ratio in T24 cells. The effects of Sakuranin on cell biological behaviors were partly annulled by Pifithrin-µ treatment. Conclusion Sakuranin suppressed T24 cell proliferation/migration/invasion, and enhanced apoptosis by potentiating autophagy through activating the p53/mTOR pathway. This study provided a theoretical basis for Sakuranin as a potential drug for clinical treatment of BC.