Zinc finger protein 24 (ZNF24) is a conserved multifunctional transcription factor associated with tumorigenesis, but its function in bladder carcinogenesis remains unclear. Herein, the expression of ZNF24 was decreased in bladder cancer (BC) cells and tissues, and patients with higher expression of ZNF24 had a better prognosis. Doxycycline-induced overexpression and knockdown of ZNF24 identified its anti-proliferative and anti-metastasis role in BC in vitro and in vivo. The potential genes for the anti-cancer role of ZNF24, involving transcriptional regulation of several factors, such as dual-specificity phosphatase 1 and squalene epoxidase. E2 conjugating enzyme UBC9 and small ubiquitin-like modifier (SUMO) 1 were found to interact with ZNF24, suggesting that ZNF24 may be SUMOylated. Consistent with the expression, ZNF24 SUMOylation levels were decreased in BC cells and tissues. Pan-SUMOylation inhibition promoted protein degradation of ZNF24. UBC9 SUMOylated ZNF24 at Lys-27 (K27) site with SUMO1 modification and the K27 mutation of ZNF24 greatly damaged the protein stability of ZNF24. Cullin 3 (CUL3), a E3 ubiquitin ligase, was responsible for the degradation of ZNF24. ZNF24 SUMOylation prevented CUL3-mediated protein degradation of ZNF24. Overall, the crosstalk between the SUMOylation and ubiquitination of ZNF24 may be a novel regulatory mechanism to block tumorigenesis and development of BC.