Background: The main treatment for overactive bladder (OAB) is the use of anticholinergic drugs initially believed to inhibit the effect of parasympathetic acetylcholine (ACh) on the detrusor; however, there is now evidence to suggest that anticholinergic drugs could interact with sensory pathways. Objective: Investigate the role of muscarinic receptors and ACh in modulating bladder afferent sensitivity in the mouse. Design, setting, and participants: Bladder and surrounding tissue were removed from wild-type male mice, placed in a recording chamber, and continually perfused with fresh oxygenated Krebs solution at 35 °C. Bladders were cannulated to allow infusion and intravesical pressure monitoring, and afferent nerve fibres innervating the bladder were dissected and put into a suction electrode for recording. Measurements: Multiunit afferent activity and intravesical pressure were recorded at baseline and during bladder distension. Experiments were conducted in the presence of muscarinic agonists and antagonist or in the presence of the cholinesterase inhibitor physostigmine. Results and limitations: Blocking muscarinic receptors using atropine (1 μM) had no effect on spontaneous afferent discharge, the afferent response to bladder distension, or on bladder compliance. However, stimulation of muscarinic receptors directly using bethanechol (100 μM) and carbachol (100 μM) or indirectly using physostigmine (10 μM) significantly inhibited the afferent response to bladder distension and concurrently reduced bladder compliance. Furthermore, prior application of nifedipine prevented the changes in bladder tone but did not prevent the attenuation of afferent responses by bethanechol or physostigmine. Conclusions: These data indicate that stimulation of muscarinic receptor pathways can depress sensory transduction by a mechanism independent of changes in bladder tone, suggesting that muscarinic receptor pathways and ACh could contribute to normal or pathologic bladder sensation.