The purpose of the study is to explore the function of P2X3 and NK1 receptors antagonists on cyclophosphamide (CYP)-induced cystitis in rats. Sixty female Sprague-Dawley (SD) rats were randomly divided into three groups. The rats in the control group were intraperitoneally (i.p.) injected with 0.9 % saline (4 ml/kg); the rats in the model group were i.p. injected with CYP (150 mg/kg); and the rats in the intervention group were i.p. injected with CYP with subsequently perfusion of bladder with P2X3 and NK1 receptors' antagonists, Suramin and GR 82334. Spontaneous pain behaviors following the administration of CYP were observed. Urodynamic parameters, bladder pressure-volume curve, maximum voiding pressure (MVP), and maximum cystometric capacity (MCC), were recorded. Pathological changes in bladder tissue were observed. Immunofluorescence was used to detect the expression of P2X3 and NK1 receptors in bladder. Cyclophosphamide treatment increased the spontaneous pain behaviors scores. The incidence of bladder instability during urine storage period of model group was significantly higher than intervention group (chi (2) = 7.619, P = 0.007) and control group (chi (2) = 13.755, P = 0.000). MCC in the model group was lower than the control and intervention groups (P < 0.01). Histological changes evident in model and intervention groups rats' bladder included edema, vasodilation, and infiltration of inflammatory cells. In model group, the expression of P2X3 receptor increased in urothelium and suburothelium, and NK1 receptor increased in suburothelium, while the expression of them in intervention group was lower. In CYP-induced cystitis, the expression of P2X3 and NK1 receptors increased in urothelium and/or suburothelium. Perfusion of bladder with P2X3 and NK1 receptors antagonists ameliorated the bladder function.