Objective To determine the median effective dose (ED50) of alfentanil for inhibiting motor responses in patients undergoing colonoscopy when combined with propofol. Methods Twenty‑five patients, men or women, aged 18‒65 years, body mass index (BMI) 18‒25 kg/m2, American Society of Anesthesiologists (ASA) physical status Ⅰ or Ⅱ, who underwent elective painless colonoscopy were selected. The patients were intravenously infused with alfentanil, with an initial dose of 5 μg/kg, before intravenous administration of propofol at a dose of 2.0 mg·kg−1·min−1. Colonoscopy was performed when the bispectral index (BIS) was 65. The dose of alfentanil in the next patient was determined according to Dixon's up‑and‑down method. The dose of alfentanil was recorded. Their mean artery pressure (MAP), heart rate, pulse oxygen saturation (SpO2), and adverse reactions were recorded when the patients breathing calmly in the room (T0), when the BIS value was 65 after administration (T1), when the colonoscope passed through sigmoid-descending colon junction (T2), when the colonoscope was withdrawn after arriving at the ileocecal valve (T3), and when the patient's eyes were open (T4). The ED50, 95% effective dose (ED95) and 95% confidence interval (CI) of alfentanil for inhibiting motor responses undergoing colonoscopy were calculated by probit regression. Results Compared with those at T0, MAP decreased at T1‒T4 (P<0.05), heart rate decreased at T1‒T3 (P<0.05), and SpO2 decreased at T2 (P<0.05). Two (8%) patients complained of mild injection pain, 1 (4%) patient developed bradycardia, 6 (24%) patients developed hypotension, and none of the patients had muscle stiffness, abdominal pain, or postoperative nausea and vomiting. The ED50 and 95%CI of alfentanil when combined with propofol for inhibiting motor responses in patients undergoing colonoscopy was 4.968 (4.488‒5.376) μg/kg, while the ED95 and 95%CI was 5.784 (5.376‒8.698) μg/kg. Conclusion The ED50 and ED95 of alfentanil combined with propofol for inhibiting motor responses in patients undergoing colonoscopy was 4.968 and 5.784 μg/kg respectively.