In order to clarify the functional role of the isolectin B4 (IB4)-binding afferent pathway in the micturition reflex, we investigated the effects on bladder activity of intrathecal application of the IB4–saporin conjugate, a targeting cytotoxin that destroys neurons binding IB4. In rats, IB4–saporin (2.5 μM) or vehicle was administered through an intrathecal catheter implanted at the level of the L6–S1 spinal cord. Three weeks after IB4–saporin administration, cystometry in conscious animals revealed a reduction in bladder overactive responses induced by intravesical capsaicin or ATP infusion without affecting normal voiding function. In histochemical studies, double staining for IB4 and saporin was detected in L6 dorsal root ganglia (DRG) neurons 2 days after the treatment. Three weeks after the treatment, the area in lamina II of the L6 spinal cord stained with IB4 was significantly reduced compared with the area stained in control rats. The staining in the L1 spinal cord was not affected. The percentage of neurons in the L6 DRG intensely labeled with IB4 was also reduced in IB4–saporin-treated rats. These results indicate that intrathecal treatment with the IB4–saporin conjugate at the level of L6–S1 spinal cord, which reduces IB4 afferent nerve terminal staining in lamina II of the L6 spinal cord as well as the number of IB4-binding neurons in L6 DRG, suppressed bladder overactivity induced by bladder irritation without affecting normal micturition. Thus targeting IB4-binding, non-peptidergic afferent pathways sensitive to capsaicin and adenosine 5′-triphosphate may be an effective treatment for overactivity and/or pain responses in the bladder.