Objective To evaluate the effects of single spay of L-menthol (NPO-11) on suppressing gastric peristalsis during upper gastrointestinal endoscopy and the influencing factor. Methods This study was a multicenter, randomized, double-blind, placebo-parallel controlled study. The eligible patients were randomly divided into two groups by randomized blocks. Patients received local spray of either NPO-11 (160 mg L-menthol) or placebo 20 mL during upper gastrointestinal endoscopy. The gastric peristalsis was recorded and evaluated before, 2 minutes after and at the end of endoscopy. The complexity of the procedure was evaluated by the researchers. The influencing factors for antiperistaltic effect of NPO-11 were analyzed. Results A total of 220 patients were enrolled from five research centers. There were 109 cases in the NPO-11 group and 111 cases in the placebo group. The baseline data of the two groups were similar and comparable. The proportion of patients with grade 1 peristalsis at 2 minutes after the treatment and at the end of endoscopy was significantly higher in the NPO-11 group than that in the placebo group [40.37% (44/109) VS 16.22% (18/111), χ2=15.93, P<0.001]. Compared with the placebo group, the proportions of weak peristalsis (grade 1 and 2) were higher in the NPO-11 group at 2 minutes after the treatment [67.89% (74/109) VS 46.85% (52/111)] and at the end of endoscopy [79.82% (87/109) VS 48.65% (54/111)]. Subgroup analysis showed that the inhibitory effect of NPO-11 on gastric peristalsis was more significant in Helicobacter pylori antibody positive group. Conclusion Local spray of NPO-11 can effectively inhibit gastric peristalsis during upper gastrointestinal endoscopy, and its effect is more significant in Helicobacter pylori antibody positive group. It could be recommended for no obvious adverse reactions , its safety, and the convenient procedure.