Purpose <p>Substance P (SP) is important in the physiological and pathophysiological functions of the bladder, including bladder sensory disorders like interstitial cystitis/bladder pain syndrome (IC/BPS) and overactive bladder (OAB). However, the impact of SP on sensory function is not fully understood. The aim of this study was to determine the effects of SP on bladder function in relation to capsaicin, specifically on bladder afferent nerve activity.</p> Methods <p>We used female Sprague–Dawley rats. The in vitro organ bath study assessed SP-induced detrusor contraction in intact and inside-out preparations of isolated whole bladders. In vivo cystometry and multi-unit bladder afferent nerve activity were measured before and after intravesical SP-instillation. Immunohistochemistry was used to locate the neurokinin-1 receptor (NK1R), an SP receptor, and transient receptor potential vanilloid 1 (TRPV1), a capsaicin receptor, in the urinary bladder.</p> Results <p>The organ bath study revealed that the intravesical pressure significantly increased in the intact preparation, but not in the inside-out preparation, by administering SP after capsaicin-desensitization. During intravesical SP-instillation, cystometric parameters remained unchanged, but bladder afferent nerve activities were significantly increased. Immunohistochemistry revealed that NK1R and TRPV1 were expressed differently throughout the bladder, but some of the expression was colocalized in the lamina propria.</p> Conclusion <p>SP caused detrusor contraction via the smooth muscle and increased bladder afferent activity via the urothelium. SP may influence such bladder function on its own rather than as a result of capsaicin exposure. These findings may shed new light on SP’s pathophysiological roles and therapeutic strategies for IC/BPS and OAB.</p>