Sialyltransferase (ST) and sialidase (SA) are the main enzymes responsible for the addition or removal of sialic acids to the terminal of glycan on glycoprotein or glycolipids. In our study, the expression of sialic acids, sialidases and sialyltransferases in normal bladder epithelial cell line HCV29, and two bladder cancer cell lines KK47 and YTS-1 were detected. Our results showed YTS-1 cells, the highly invasive cell line, expressed higher level of sialic acids than other two cell lines. However, the level of Neu1 in YTS-1 cells was the lowest among all the cell lines. The expression of Toll like receptors TLR1,2,3,4 was consistent with neu1 expression in these three cell lines. When normal bladder cell HCV29 underwent TGF-β induced epithelial to mesenchymal transition (EMT) process, expression of Neu1 and TLR3 were significantly decreased. When Neu1 expression was inhibited in HCV29 cell line, the level of TLR3 was also reduced. Overexpression of Neu1 in YTS-1cells resulted in TLR3 increase accompanied with the activation of NF-κB signaling pathway. Our results indicated that Neu1 shared closed relationship with the expression of TLRs in bladder cancer cells, which may potentially provide therapeutic candidates in treating bladder cancer.