Bladder cancer is the most common malignant tumor of the urinary tract. In this study, 90 lupane triterpene derivatives, previously synthesized in the laboratory, were systematically evaluated for their potential effects against bladder cancer by cytotoxicity screening against five urinary tumor cell lines. Bioinformatics and molecular dynamics methods were used to investigate the mechanism of action of compound 27 in depth. Most of the derivatives effectively inhibited tumor cell growth, and structure-activity relationship analysis revealed that introducing an indole moiety significantly enhanced the biological activity. The peak activity was reached when the dibromoalkyl chain length was C = 5 (IC = 1.121 μM). By integrating transcriptomic data and TCGA findings, we identified 11 key targets, among which DUSP5 and SCG2 showed significant differential expression. Further analysis revealed meaningful insights into the clinical association, 10-year survival prognosis, and immune infiltration. The present study further clarified the effects of compound 27 on the expression of DUSP5 and SCG2 in tumor cells after treatment by a combination of RNA-seq and RT-qPCR. Molecular docking confirmed the stable binding of compound 27 to DUSP5, which was confirmed by molecular dynamics simulations. Compound 27 inhibited bladder cancer progression by upregulating DUSP5 expression and negatively regulating the p38 MAPK pathway, modulating the immune response and promoting apoptosis.