Abstract Background and purpose: Several studies have indicated that reactive oxygen species (ROS) can lead to detrusor overactivity (DO), but the underlying mechanisms are not known. Hydrogen dioxide (H2O2) is used commonly to investigate the effects of ROS. In present study, we aimed to investigate the effects of H2O2 on phasic spontaneous bladder contractions (SBCs) of isolated human-bladder strips (iHBSs) and the underlying mechanisms. Experimental approach: Samples of bladder tissue were obtained from 26 patients undergoing cystectomy owing to bladder cancer. SBCs of iHBSs were recorded in organ-bath experiments. Immunofluorescence staining was conducted to confirm the TRPA1 expression on sensory nerves. Key results: H2O2 (1μM–10mM) concentration-dependently increased the SBCs of iHBSs. These enhancing effects could be mimicked by an agonist of transient receptor potential (TRP)A1 channels (allyl isothiocyanate) and blocked with an antagonist of TRPA1 channels (HC030031; 10μM). H2O2 induced enhancing effects also could be attenuated by desensitizing sensory afferents with capsaicin (10μM), blocking nerve firing with TTX (1μM), blocking neurokinin effects with NK2 receptor antagonist (SR48968, 10μM), and blocking PGE2 synthesis with indomethacin (10μM), respectively. Conclusions and implications: Our study: (i) suggests activation of TRPA1 channels on bladder sensory afferents, and then release of substance P or PGE2 from sensory nerve terminals, contribute to the H2O2-induced enhancing effects on SCs of iHBSs; (ii) provides insights for the mechanisms underlying ROS leading to DO; (iii) indicates that targeting TRPA1 channels might be the promising strategy against overactive bladder in conditions associated with excessive production of ROS.