Objective To explore the role of P300/CBP-associated factor (PCAF) in the repair of human bladder carcinoma (bladder carcinoma) cell DNA damage and the maintenance of tumor stem cell stemness. Methods Human bladder cancer cell line 5637 was transfected with Ad-PCAF RNAi. The transfection effect was detected by fluorescence microscopy and Western blot. Cisplatin was selected to induce DNA damage in bladder cancer cells. The proliferative activity of the injured cells was detected by MTS. The recruitment of PCAF and γ-H2AX in injured cells was detected by immunofluorescence staining. The degree of DNA damage induced by cisplatin was analyzed by comet assay. CD44+ labeled tumor stem cells were sorted from bladder cancer cells. The expression levels of Bca stem cell markers 67LR, OCT4 and YAP1 were detected by Western blot. Cell pelletizing assay was used to detect the changes of cell stem.Results An Ad-PCAF RNAi transfected into 5637 cells significantly inhibited the expression of PCAF (P<0.05). After the PCAF expression was down-regulated, the activity of 5637 cells was decreased under 20, 40, 60, 80, 100 nmol/L cisplatin treatment, and the sensitivity of 5637 cells to cisplatin was enhanced, the formation of γ-H2AX focal point was significantly increased, the cell tail was longer, and the number and distance of trailing cells were increased (P<0.05). The bladder cancer stem cells with 93% CD44+ and positive expression of 67LR, OCT4 and YAP1 were obtained. By down-regulating the expression of PCAF, the spheroids of tumor stem cells became smaller and the number of spheroids formed was also decreased (P<0.05). Conclusion Down-regulating the expression of PCAF can inhibit DNA damage and repair of bladder cancer cells and regulate the maintenance of stemness of cancer stem cells.