Background and Purpose: There is increasing evidence suggesting that ROS play a major pathological role in bladder dysfunction induced by bladder inflammation and/or obstruction. The aim of this study was to determine the effect of H2O2on different types of bladder afferents and its mechanism of action on sensory neurons in the guinea pig bladder. Experimental Approach: ‘Close-to-target’ single unit extracellular recordings were made from fine branches of pelvic nerves entering the guinea pig bladder, in flat sheet preparations, in vitro. Key Results: H2O2(300–1000 μM) preferentially and potently activated capsaicin-sensitive high threshold afferents but not low threshold stretch-sensitive afferents, which were only activated by significantly higher concentrations of hydrogen peroxide. The TRPV1 channel agonist, capsaicin, excited 86% of high threshold afferents. The TRPA1 channel agonist, allyl isothiocyanate and the TRPM8 channel agonist, icilin activated 72% and 47% of capsaicin-sensitive high threshold afferents respectively. The TRPA1 channel antagonist, HC-030031, but not the TRPV1 channel antagonist, capsazepine or the TRPM8 channel antagonist, N-(2-aminoethyl)-N-[[3-methoxy-4-(phenylmethoxy)phenyl]methyl]thiophene-2-carboxamide, significantly inhibited the H2O2-induced activation of high threshold afferents. Dimethylthiourea and deferoxamine did not significantly change the effect of H2O2on high threshold afferents. Conclusions and Implications: The findings show that H2O2, in the concentration range detected in inflammation or reperfusion after ischaemia, evoked long-lasting activation of the majority of capsaicin-sensitive high threshold afferents, but not low threshold stretch-sensitive afferents. The data suggest that the TRPA1 channels located on these capsaicin-sensitive afferent fibres are probable targets of ROS released during oxidative stress.