Toll-like receptor 7 (TLR7) is associated with the pathophysiology of systemic lupus erythematosus and Sjögren syndrome, well-known diseases accompanying interstitial cystitis (IC). We studied TLR7 expression in the bladder of patients with Hunner-type IC (HIC) and its functional roles in bladder inflammation and nociception using mice. Bladder biopsy specimens were obtained from patients with HIC. Specimens from the noncancerous portion of the bladder of patients with bladder cancer served as controls. The specimens were examined by immunohistochemistry and real-time polymerase chain reaction of TLR7. Loxoribine (LX), a TLR7 agonist, was instilled in the bladder of C57BL/6N female mice, and TLR7-mRNA expression and histological changes of the bladder, bladder pain–like licking behavior, voiding behavior, cystometry, and bladder afferent nerve activities were investigated. The effects of hydroxychloroquine, a TLR7 antagonist, on the LX-induced changes on cystometry and voiding behavior were studied. The number of TLR7 immuno-reactive cells and the mRNA expression of TLR7 were significantly increased in HIC specimens. Intravesical instillation of LX induced edema, congestion, inflammation, and significantly increased TLR7-mRNA expression in the mouse bladder. Loxoribine-instillation also significantly increased licking behavior, voiding frequency, and afferent nerve activities associated with decreased single-voided volume and intercontraction interval of micturitions. Hydroxychloroquine reversed the LX-induced cystometric and voiding behavioral changes. Toll-like receptor 7 was up-regulated in the bladder mucosa of patients with HIC, and activation of TLR7 in the mouse bladder induced cystitis with sensory hyperactivity of the bladder. Blocking the TLR7 pathway may be an innovative treatment target of HIC.