Abstract: Objective To observe the effect of S100A4 gene silencing mediated by small interfering RNA (siRNA) on the proliferation of bladder cancer stem cells (CSCs) and their capacity of xenograft tumor formation. Methods MB49 bladder cancer stem cells (MCSCs) were isolated and identified. The differentially expressed protein S100A4 was identified in MCSCs using isobaric tags for relative and absolute quantitation technology (iTRAQ). A siRNA targeting S100A4 was constructed and transfected into MCSCs, and its inhibitory effects on S100A4 expression in MCSCs were assessed with Western blotting and qPCR. The effects of siRNA-mediated S100A4 silencing on the proliferation and xenograft tumor formation ability of MCSCs were observed. Results Among the 65 differentially expressed proteins identified by iTRAQ combined with LC/MS/MS, S100A4 protein showed the most distinct differential expression in MCSCs. Transfection of MCSCs with S100A siRNA significantly inhibited the expressions of S100A4 at both mRNA and protein levels, caused obvious suppression of the cell proliferation, and attenuated the xenograft tumor formation ability of the cells in nude mice. Conclusion S100A4 in MCSCs is associated with the recurrence and metastasis of bladder cancer. S100A4 may serve as a potential therapeutic target for eliminating bladder CSCs.