Objective To study whether botulinum toxin type A (BTX-A) can inhibit the spontaneous and acetylcholine (ACh)- or substance P (SP)-induced contraction of rat lower esophageal sphincter in vitro, and to discuss the related mechanism. Methods The lower esophagus muscle strips were taken from Sprague-Dawley rats and were randomly divided into control group, BTX-A group, ACh group, ACh+BTX-A group, ACh+Atropine group, SP group, SP+APTL-SP group and SP+BTX-A group. The contractile graph of the muscle strips was recorded by physiological experimental system Biolap-420E. Results BTX-A significantly decreased the spontaneous contractile tension and amplitude in the lower esophageal sphincter(P<0.05). ACh significantly enhanced the contractile tension and amplitude in the lower esophageal sphincter (P<0.01), which could be significantly inhibited by both BTX-A and Atropine (P<0.01). SP significantly enhanced the contractile tension in the lower esophageal sphincter (P<0.01), which could be significantly inhibited by both BTX-A and APTL-SP (P<0.01). Conclusion ACh and SP can enhance the spontaneous contractility of lower esophageal sphincter. BTX-A can inhibit ACh- and SP-induced enhancement of lower esophageal sphincter contraction.