In this study, we used a well-established animal model to investigate changes in the peptidergic and parasympathetic innervation of the bladder following chronic bladder inflammation. Adult female Sprague–Dawley rats were injected with either 70 mg/kg cyclophosphamide diluted in saline, i.p., once every 3 days or saline. After 10 days, all animals were tested for urinary frequency and number of low volume voids, as well as symptoms of spontaneous pain. At the end of 12 days, all animals were perfused with histological fixatives and the urinary bladders processed for immunofluorescence using antibodies against calcitonin gene-related peptide and the vesicular acetylcholine transporter as markers, respectively, of peptidergic primary afferent fibers and parasympathetic efferent fibers. We show that animals treated with cyclophosphamide had inflamed bladders and displayed high urinary frequency as well as some indicators of spontaneous pain, such as piloerection and a rounded-back posture. Furthermore, they had a significant increase in the density of both parasympathetic and peptidergic sensory fibers in the bladder mucosa and an increase in peptidergic sensory fibers in the detrusor muscle. Based on these results, we suggest that peripheral sprouting of parasympathetic and peptidergic fibers could be a mechanism responsible for sensitization of the bladder, leading to urinary symptoms. Since we observed that the parasympathetic and peptidergic fibers often wrapped around one another and that their varicosities were very close, these two fiber populations may be interacting with each other to lead to and maintain sensitization. Future studies are required to establish the role of this fiber sprouting in bladder symptoms.