Background & Aims: Substance P (SP) is commonly co-expressed with acetylcholine in enteric motor neurons and according to the classical paradigm, both these neurotransmitters excite smooth muscle via parallel pathways. We hypothesized that in addition, SP was responsible for maintaining the muscular responsiveness to acetylcholine (ACh). We tested this hypothesis by using botulinum toxin (BoNT/A), a known blocker of vesicular release of neurotransmitters including acetylcholine and neuropeptides. Methods: BoNT/A was injected into rat pyloric sphincter in different doses; as control we used boiled BoNT/A. At the desired time point, the rats were sacrificed and pyloric contractility was measured ex vivo in an organ bath and by measuring phosphorylation of myosin light chain 20 (MLC₂₀). Results: BoNT/A (10 IU) significantly reduced the response of pyloric muscle to exogenous ACh, an effect that was accompanied by reduced MLC₂₀ phopshorylation in the muscle. Both effects were reversed by exogenous SP. CP-96345, a NK1 receptor antagonist, blocked the ability of exogenous SP to reverse the cholinergic hyporesponsiveness as well as the reduction in MLC₂₀ phosphorylation induced by BoNT/A. Conclusion: We have identified a novel role for SP as a co-neurotransmitter that appears to be important for the maintenance of muscular responsiveness to the principal excitatory neurotransmitter, acetylcholine. These results also provide new insight into the effects of botulinum toxin on the enteric nervous system and gastrointestinal smooth muscle.