Background and Aims Ultrasound-guided popliteal vein puncture is one of the common approaches for the intravenous intervention of the lower limb veins. At present, the prone position is frequently used for this procedure. However, long-time prone-positioned surgery is uncomfortable for patients. Therefore, this study was performed to investigate the feasibility of ultrasound-guided popliteal vein puncture in the supine position and compare the advantages and disadvantages of supine and prone position.Methods Patients with chronic venous disease were randomly assigned to two groups and underwent ultrasound-guided popliteal vein puncture in a supine or prone position, respectively. The variables, such as the operative time for popliteal vein puncture and visual analog score (VAS) for the degree of discomfort of patients and the incidence of postoperative complications, were analyzed.Results A total of 27 patients with iliac vein stenosis were enrolled, including 14 cases (16 limbs) in the supine position group and 13 cases (16 limbs) in the prone position group. There were no significant differences in the baseline data and the treatment methods of the affected veins between the two groups of patients (all P>0.05). The operative time for popliteal vein puncture showed no significant difference between the supine position group and prone position group [3.7 (3.4-6.2) min vs. 4.2 (3.5-4.4) min, P>0.05]. The VAS value was significantly lower in supine position group than that in prone position group [2.0 (1.0-2.8) vs. 6.0 (4.0-8.0), P<0.01]. Bleeding from a branch of the popliteal artery occurred in one supine position group case, which was successfully cured by ultrasound-guided compression of the lesion.Conclusion Popliteal vein puncture in the supine position does not prolong the operative time compared with prone position, but it can significantly reduce the discomfortable suffering of patients. So, it is recommended to be used in clinical practice.