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Objective: Brucein D (BrD), a quassinoid isolated from Brucea javanica fruit, reportedly demonstrates anti-cancer activity. This study's objective is to evaluate the cytotoxicity of Brucein D and its ability to induce apoptosis in T24 bladder cancer cells., Methods: We investigated the cytotoxic activity of BrD against the T24 cell through the induction of apoptosis in vitro. This cytotoxic activity was evaluated with ΜΤΤ assay and followed by Calcein-AM/PI viability staining. Apoptotic activity was determined with Hoechst 33342 nuclear staining and DNA fragmentation. Doxorubicin and docetaxel were used as a positive control. Evaluation of apoptotic-related gene expression, Bax, Bak, Bcl2, and p53 was also performed using semi-quantitative PCR analysis. Statistical analysis was conducted using One-way ANOVA followed by post hoc test Turkey's HSD (Honestly Significance Difference)., Results: Results show that BrD had high toxicity against T24 bladder cancer cells with an IC50 value of 7.65 ± 1.2 µg/mL but relatively less toxic to 1BR3 normal skin fibroblast cells compared to the doxorubicin and docetaxel treated cells. The viability assay shows that BrD significantly increases the percentage of dead cells relative to control in a dose-dependent manner. Furthermore, the percentage of cells with apoptotic appearance was significantly higher in group treated with BrD IC50 (56.04±3.09%) compared to control (9.42±2.88). The result was similar to doxorubicin IC50 (58.97±12.31) but lower than docetaxel IC50 (74.42±9.79). DNA fragmentation in gel electrophoresis was also observed in T24 cells treated with BrD. Apoptosis was also verified by an alteration in the expression of apoptosis-related genes, upregulation of Bax, Bak, and p53, and downregulation of Bcl-2., Conclusion: BrD has shown a cytotoxic effect against T24 bladder cancer cells. Hence, it is a promising natural compound for the management of bladder cancer by induction of apoptosis through activation of the intrinsic pathway, with low toxicity to normal cells.