Ketamine abusers may develop ulcerative cystitis and severe lower urinary tract symptoms, which is a medical dilemma. Recently, researchers have found the endemic of ketamine-induced cystitis worldwide. The intravesical administration of liposome-encapsulated onabotulinumtoxinA (Lipotoxin) might facilitate the healing of the damaged urothelium from liposomes, and reduce the urinary symptoms by onabotulinumtoxinA-induced chemo-denervation. Using female Sprague-Dawley rats, we investigated the effects of Lipotoxin on ketamine-induced cystitis. Functional magnetic resonance imaging, metabolic cage study, and cystometry were conducted. Paraffin-embedded sections were stained. The bladder mucosa and muscle proteins were assessed through Western blotting. We observed that repeated intravesical Lipotoxin instillation could improve suburothelial hemorrhage, recover the urothelial tight junction and adhesion proteins (zonula occludens-1 and E-cadherin), ensure less substance P in the urothelium, inhibit the overexpression of inflammatory mediators (IL-6, TNF-α, nuclear NF-κB, and COX-2) in the detrusor, suppress the upregulation of the mucosal TRPV1 and detrusor M2-mAChR, and ameliorate bladder overactivity in the ketamine-treated rats. These data reveal the mechanisms underlying the action of Lipotoxin in ketamine-induced cystitis of rats, which provide a basis of Lipotoxin for further treating ketamine-induced cystitis in humans.