We examined bladder and urethral sphincter activity in mice with or without spinal cord injury (SCI) after C-fiber afferent desensitization induced by capsaicin pretreatment and changes in electrophysiological properties of mouse bladder afferent neurons 4 weeks after SCI. Female C57BL/6N mice were divided into 4 groups; (1) spinal intact (SI)-control, (2) SI-capsaicin pretreatment (Cap), (3) SCI-control and (4) SCI-Cap groups. Continuous cystometry and external urethral sphincter (EUS)-electromyogram (EMG) were conducted under an awake condition. In Cap groups, capsaicin (25, 50 or 100 mg/kg) was injected subcutaneously 4 days before experiments. In SI-Cap group, 100 mg/kg capsaicin pretreatment significantly increased bladder capacity and decreased the silent period duration of EUS-EMG compared to SI-control group. In SCI-Cap group, 50 and 100 mg/kg capsaicin pretreatment respectively decreased the number of non-voiding contractions (NVCs), and the duration of reduced EUS activity during voiding compared to SCI-control group. In SCI mice, hexamethonium, a ganglionic blocker, almost completely blocked NVCs, suggesting that they are of neurogenic origin. Patch clamp recordings in capsaicin-sensitive bladder afferent neurons from SCI mice showed hyperexcitability evidenced by decreased spike thresholds and increased firing rate compare to SI mice. These results indicate that capsaicin-sensitive C-fiber afferent pathways, which become hyperexcitable after SCI, can modulate bladder and urethral sphincter activity in awake SI and SCI mice. Detrusor overactivity as shown by NVCs in SCI mice is significantly, but partially, dependent on capsaicin-sensitive C-fiber afferents; whereas the EUS relaxation during voiding is enhanced by capsaicin-sensitive C-fiber bladder afferents in SI and SCI mice.