Functional disorders (i.e., interstitial cystitis/painful bladder syndrome and irritable bowel syndrome) are associated with hyperexcitability of afferent nerves innervating the urinary tract and the bowel respectively. Various non-5-HT receptor mRNA transcripts are expressed in mouse urothelium and exert functional responses to 5-HT. Whilst 5-HT receptors were not detected in mouse urothelium, 5-HT receptors expressed on bladder sensory neurons plays a role in bladder afferent excitability under both normal conditions and in a mouse model of chronic visceral hypersensitivity (CVH). These data suggest that the role 5-HT receptors play in bladder afferent signaling warrants further study as a potential therapeutic target for functional bladder disorders.Serotonin (5-HT) is an excitatory mediator, which in the gastrointestinal (GI) tract, plays a physiological role in gut-brain signaling and which is dysregulated in functional GI disorders such as irritable bowel syndrome (IBS). Patients suffering from IBS frequently suffer from urological symptoms characteristic of interstitial cystitis/painful bladder syndrome, which manifests due to cross-sensitization of shared innervation pathways between the bladder and colon. However, a direct modulatory role of 5-HT in bladder afferent signaling and its role in colon-bladder neuronal crosstalk remain elusive. The aim of this study was to investigate the action of 5-HT on bladder afferent signaling in normal mice and mice with chronic visceral hypersensitivity (CVH) following trinitrobenzenesulfonic acid (TNBS) induced colitis. Bladder afferent activity was recorded directly using ex vivo afferent nerve recordings. Expression of 14 5-HT receptor subtypes, the serotonin transporter (SERT) and 5-HT producing enzymes were determined in the urothelium using RT-PCR. Retrograde labelling of bladder projecting dorsal root ganglion (DRG) neurons was used to investigate expression of 5-HT receptors using single cell RT-PCR, while sensory neuronal and urothelial responses to 5-HT were determined by live cell calcium imaging. 5-HT elicited bladder afferent firing predominantly via 5-HT receptors expressed on afferent terminals. CVH animals showed a downregulation of SERT mRNA expression in urothelium, suggesting increased 5-HT bioavailability. Granisetron, a 5-HT antagonist, reversed bladder afferent hypersensitivity in CVH mice. These data suggest 5-HT exerts a direct effect on bladder afferents to enhance signaling.5-HT antagonists could therefore be a potential therapeutic target to treat functional bladder and bowel disorders. This article is protected by copyright. All rights reserved.