The role of adipose-derived stromal stem cells (ADSCs) in BLCA progression is unclear. We investigated the effects of invasion, stemness, Epithelial-mesenchymal transition (EMT), and drug resistance of BLCA cells co-cultured with ADSCs for a long period of time. Cells were divided into six groups: ADSCs group, ADSCs: T24 group (10:1, 3:1 and 1:1 groups), ADSCs-derived conditioned medium group (CM) and T24 cell group (T24), and cells in each group were cultured to 14 days, and puromycin (puro) was added to the co-cultured cell to remove ADSCs cells without puro resistance, and then the function of T24 cells (10:1-COC, 3:1-COC and 1:1-COC) after co-culture was studied; CCK-8 assay, Transwell, Wound healing, Flow cytometry, RNA-sequencing, qRT-PCR and Western Bloting assay were used to detect cell proliferation, invasion, migration, apoptosis and cellular mRNA and protein expression levels, respectively. We unexpectedly found enhanced stemness and drug resistance of BLCA cells after prolonged contact culture with ADSCs. T24 cells after co-culture mediated cell proliferation, invasion, EMT, stemness, drug resistance and immune escape by up-regulating MDM2, mt-P53 and PD-L1, compared to CM and T24 groups. The inhibitor Atezo and CP-31,398 eliminated mt-P53 and PD-L1-mediated T24 cell drug resistance and stemness, respectively. This study demonstrated that after prolonged co-culture of BLCA cells with ADSCs, the stemness, drug resistance, and immune evasion of T24 cells were dramatically enhanced, suggesting that long-term resident ADSCs in the bladder cancer tumor microenvironment play a procarcinogenic role.