Interstitial cystitis/bladder pain syndrome (IC/BPS) is a type of chronic bladder inflammation characterized by increased voiding frequency, urgency and pelvic pain. The sensitization of bladder afferents is widely regarded as one of the pathophysiological changes in the development of IC/BPS. There is evidence that adenosine A receptors are involved in regulating the sensitization of sensory afferents. However, the effect of adenosine A receptors on cystitis remains unknown. In the present study, a rat model of chronic cystitis was established by intraperitoneal injection with cyclophosphamide (CYP). Cystometry and behavioral tests were performed to investigate bladder micturition function and nociceptive pain. The rats with chronic cystitis showed symptoms of bladder overactivity, characterized by an increase in bladder voiding frequency and voiding pressure. CYP treatment significantly increased the expression of the A receptor in bladder afferent fibers and dorsal root ganglion (DRG) neurons. The A receptor antagonist ZM241385 prevented bladder overactivity and hyperalgesia elicited by CYP-induced cystitis. In addition, the A receptor and TRPV1 were coexpressed on DRG neurons. The TRPV1 antagonist capsazepine blocked bladder overactivity induced by the A receptor agonist CGS21680. In contrast, ZM241385 significantly inhibited the capsaicin-induced increase in intracellular calcium concentration in DRG neurons. These results suggest that suppression of adenosine A receptors in bladder afferents alleviates bladder overactivity and hyperalgesia elicited by CYP-induced cystitis in rats by inhibiting TRPV1, indicating that the adenosine A receptor in bladder afferents is a potential therapeutic target for the treatment of IC/BPS.