Sensitization of neuronal pathways and persistent afferent drive are major contributors to somatic and visceral pain. However, the underlying mechanisms that govern whether afferent signaling will give rise to sensitization and pain are not fully understood. In the present report, we investigated the contribution of acid-sensing ion channels (ASICs) to bladder nociception in a model of chemical cystitis induced by cyclophosphamide (CYP). We found that the administration of CYP to mice lacking ASIC3, a subunit primarily expressed in sensory neurons, generates pelvic allodynia at a time point at which only modest changes in pelvic sensitivity are apparent in wild-type mice. The differences in mechanical pelvic sensitivity between wild-type and knockout mice treated with CYP were ascribed to sensitized bladder C nociceptors. Deletion of from bladder sensory neurons abolished their ability to discharge action potentials in response to extracellular acidification. Collectively, the results of our study support the notion that protons and their cognate ASIC receptors are part of a mechanism that operates at the nerve terminals to control nociceptor excitability and sensitization. Our study indicates that protons and their cognate acid-sensing ion channel receptors are part of a mechanism that operates at bladder afferent terminals to control their function and that the loss of this regulatory mechanism results in hyperactivation of nociceptive pathways and the development of pain in the setting of chemical-induced cystitis.