Abstract: In order to investigate the effect of usnic acid on human bladder cancer, cultured human bladder cancer cells T24 and RT4 were treated with different concentrations of usnic acid (12.5, 25, 50, 100, 200 μmol/L) for 24 h and 48 h, respectively, and the proliferation of T24 and RT4 cells was detected by MTT assay. The results showed that intracellular reactive oxygen species (ROS) levels were increased after treatment with usnic acid, and apoptosis of T24 cells was induced through mitochondrial pathway and death receptor pathway: (1)Usnic acid inhibited the proliferation of T24 and RT4 cells in a time-dependent and dose-dependent manner, and the inhibitory rate of usnic acid on T24 cell proliferation was higher. (2)By Hoechst 33342 staining and Annexin V-FITC/PI double staining, apoptotic T24 cells gradually increased with the increase of the concentration of usnic acid. (3)After treatment with usnic acid, the level of intracellular ROS increased, the expression of Bax, cleaved-Caspase-8, cleaved-Caspase-9 and cleaved-Caspase-3 protein increased, the expression of Bcl-2, Caspase-3, Caspase-8 and caspase-9 decreased, and the ratio of Bax protein expression to Bcl-2 protein expression increased.