Objective To investigate the biological effects of Pasteurella multocida (Pm) culture supernatant and Pm-derived outer membrane vesicle (OMV) on bladder cancer cells. Methods Pm was cultured and its supernatant was collected. The effects of the supernatant on proliferation, migration and invasion of bladder cancer cell lines (T24 and 5637) were assessed by cell counting kit 8 (CCK-8), wound healing assay, and Transwell migration and invasion assays with phosphate-buffered saline (PBS) and brain heart infusion (BHI) broth as controls. Pm-OMV were isolated from the supernatant via ultracentrifugation, and the remaining components of the supernatant served as control. The effects of Pm-OMV on proliferation, migration and invasion of T24 and 5637 cells were assessed by CCK-8 and Transwell migration and invasion assays. Apoptosis was analyzed by flow cytometry. A nude mouse xenograft tumor model was established. After intratumoral multi-point injections of Pm-OMV or PBS, the tumor growth was evaluated and the effects of Pm-OMV on proliferation and apoptosis of bladder cancer cells in vivo were verified by Ki67 (a proliferation marker) immunohistochemical staining and TUNEL assay. Results Pm culture supernatant significantly inhibited the proliferation, invasion, and migration of T24 and 5637 cells in vitro compared with PBS and BHI controls (all P＜0.01). Pm-OMV not only inhibited the proliferation, invasion, and migration of T24 and 5637 cells, but also induced the apoptosis, and the differences were significant compared with the remaining components of the supernatant (all P＜0.05). The nude mouse subcutaneous tumor transplantation experiment further confirmed that Pm-OMV inhibited the proliferation of bladder cancer cells and promoted apoptosis in vivo, and the differences were significant compared with the PBS control (all P＜0.05). Conclusion Pm-OMV can inhibit the proliferation, invasion, and migration of bladder cancer cells and promote the apoptosis. It provides an experimental basis for studying the mechanism of microbial regulation of tumor progression and for developing new treatment strategies for bladder cancer.