Objective To investigate the effect of Brazilin on bladder cancer and its possible mechanism through in vitro and in vivo studies. Methods The proliferation inhibitory effect of Brazilin on T24 cells and T24 cells overexpressing c-Fos ( T24-c-Fos cells ) was detected by the MTT method. The effects of Brazilin on the migration and colony formation ability of the two types of cells were detected by cell scratch and colony formation assays. The effects of Brazilin on the expression of c-Fos, TNFAIP3 ( Tumor Necrosis Factor Alpha-Induced Protein 3 ) and NF-κB in the two types of cells were detected by qPCR and Western blot at the mRNA and protein levels, respectively. T24 cells and T24-c-Fos cells bladder orthotopic transplanted tumor mice models were constructed, Brazilin intervention, after 60 days, the bladder weights were dissected and recorded, HE staining was used to observe the morphology of orthotopic tumors of bladder, the expression of c-Fos, TNFAIP3 and NF-κB in the orthotopic tumors of bladder were detected by immunohistochemistry, qPCR and Western blot. Results Brazilin inhibited the proliferation, migration and colony formation ability of T24 cells and T24-c-Fos cells (P<0.05), compared with T24 cells, the inhibitory effect on T24-c-Fos cells were significantly increased (P<0.05). Brazilin upregulated the expressions of c-Fos and TNFAIP3 in T24 cells and T24-c-Fos cells at both mRNA and protein levels, downregulated the expression of NF-κB, there were statistically significant difference compared with the corresponding blank control group (P<0.05), compared with T24 cells, the degree of change of the above indicators were more significant in T24-c-Fos cells (P<0.05). The mice models of orthotopic bladder transplant tumor with T24 cells and T24-c-Fos cells were successfully constructed, and the bladder weight of the Brazilin intervention groups were lower than that in the corresponding model groups (P<0.05). The inhibitory effect of Brazilin on orthotopic bladder transplant tumors bearing T24-c-fos cells were significantly higher than that bearing T24 cells (P<0.05). HE staining results showed that the bladder structure disappeared in all experimental groups, with densely packed tumor cells exhibiting nuclear atypia and a high nuclear-to-cytoplasmic ratio, in the T24+Brazilin group and T24-c-Fos+Brazilin group, a large number of necrotic cells were observed, with nuclear pyknosis or karyolysis. The results of immunohistochemistry, qPCR, and Western blot showed that, Brazilin promoted the expression of c-Fos and TNFAIP3, and decreased the expression of NF-κB in orthotopic bladder transplant tumors, compared with bearing T24 cells, the changes of the above indicators in orthotopic bladder transplant tumors bearing T24-c-fos cells were more significant (P<0.05). Conclusion Brazilin may upregulate TNFAIP3 expression through c-Fos, thereby inhibiting the NF-κB signaling pathway and exert anti-bladder cancer effects.