Melatonin is a circadian rhythm regulator capable of controlling a variety of physiological processes in the body. It predominantly acts via the melatonin 1 (MT1) and MT2 receptors expressed in the CNS neurons and peripheral organs and tissues. Melatonin can modulate urinary bladder function, however, to date it is not known if melatonin can regulate activity of sensory neurons innervating the bladder. Bladder afferents play an important role in urine storage and voiding. Therefore, this study aims to determine if melatonin can regulate mechanosensitivity of 2 major classes of sensory neurons in the guinea pig bladder: stretch-insensitive mucosal and low threshold stretch-sensitive muscular-mucosal afferents. The effects of melatonin on the mechanosensitivity of mucosal and muscular-mucosal afferents were measured ex vivo using single unit extracellular recording. Melatonin did not affect the responses of mucosal afferents to stroking of their receptive fields but did concentration-dependently, significantly inhibit 69% of muscular-mucosal afferents responses to stroking and bladder stretch. This inhibitory effect was not affected by the MT1 receptor antagonist, S26131 but was blocked by the selective MT2 receptor antagonists, K-185 and 4-P-PDOT. Forskolin significantly potentiated the responses of muscular-mucosal afferents to stroking and stretch, which were prevented by melatonin. These findings demonstrate a direct inhibitory effect of melatonin on the mechanosensitivity of low threshold stretch-sensitive muscular-mucosal bladder afferents acting via MT2 receptors, which is independent from its action on detrusor muscle. This may have important clinical implications for the treatment of many common bladder disorders including nocturia.