Background: Intestinal spasm and peristalsis during colonoscopy are common but undesirable phenomena, which can easily lead to a missed diagnosis of colorectal polyps and other diseases, and antispasmodic drugs can have adverse side effects. Previous studies find that acupuncture can regulate abnormal gastrointestinal motility. But evidence quality is low and limited at present, and high-quality studies are required. So this study sought to explore the efficacy and safety of acupuncture in inhibiting colonic spasm during endoscopy. Methods: In this prospective, single-blinded, randomized controlled trial, 54 patients experiencing intestinal spasms during colonoscopy were randomly assigned to receive either acupuncture of the bilateral Hegu (LI 4) and Neiguan (PC 6) points (n=27) or sham acupuncture (n=27). The sham points were located 1 cm above the proximal end of the true points and had no known function. The primary outcome was the latency time to colonic spasm suppression, and the secondary outcomes were the duration of colonic spasm suppression, the proportion of patients with rebound spasms within 5 minutes, and adverse events related to acupuncture-related side effects. Results: A total of 54 patients were eligible, and 27 in each group. There was no significant difference in the background characteristics of the patients in the 2 groups. The latency time to spasm suppression of the treatment group was significantly shorter than that of the sham control group (acupuncture: 32.00 s vs. sham: 82.00 s; P<0.001). However, the duration of colonic spasm suppression was similar (acupuncture: 300 s vs. sham: 268 s; P=0.142). No rebound spasms were observed in the treatment group but rebound spasms were observed in 3 patients in the sham control group (acupuncture: 0% vs. sham: 11.1%; P=0.236). No adverse events were observed in either group. Conclusions: Acupuncture of the bilateral Hegu (LI 4) and Neiguan (PC 6) points can shorten the latency time to spasm suppression, and may be used to suppress colonic spasm during colonoscopy. Trial registration: Chinese Clinical Trial Registry ChiCTR2000037796