Abstract: Bladder cancer is one of the most common malignancies, whose tumorigenesis have not been elucidated. We developed two novel monoclonal antibody BCMab1 and BCMab8, which could separate bladder cancer stem cells（BCSCs）.We found that Hedgehog signaling was activated in BCSCs. We also found that glycosyl transferase GALNT1 was highly expressed in BCSCs, which deduced that glycosylation modification mediated by GALNT1 participated in activation of Hedgehog signaling. We will identify the new surface markers in BCSCs, and verify their biological characteristics. Through the single cell sequencing, we will determine the origin of the BCSCs. We will explore the effects of Hedgehog signaling on self-renewal of BCSCs. We will elucidate SHh glycosylation modification mediated by GALNT1 is required for activation of SHh protein, and verify the Hedgehog signaling mediated by GALNT1 plays the most important role in maintaining self-renewal for BCSCs. We will identify the peptide inhibitors of GALNT1 and investigate its intervention effect on self-renewal of BCSCs. Our study will reveal the mechanism of self-renewal for BCSCs regulated by Hedgehog signaling, and provide theoretical foundations for designing drugs, which suppress self-renewal of BCSCs.