Objective <p>The study aimed to compare the surgical efficacy and safety of hysterectomy by robot-assisted single-port transvaginal natural orifice transluminal endoscopic surgery (RvNOTES), robot-assisted transumbilical single-port laparoscopic surgery (RTSP-LS), and conventional single-port laparoscopic-assisted vaginal hysterectomy (SP-LVH).</p> Methods <p>In this study, we retrospectively analyzed clinical data of 117 patients who underwent robot-assisted vNOTES hysterectomy (RvNOTES-H), robot-assisted transumbilical single-port laparoscopic hysterectomy (RTSP-LH), and conventional single-port laparoscopic-assisted vaginal hysterectomy (SP-LVH) between September 2019 and December 2023 at the Department of Gynecology, the First Affiliated Hospital of Zhengzhou University, China. Clinical characteristics and surgical outcomes were collected and analyzed.</p> Results <p>All surgeries were successfully completed as planned. No significant differences were observed in the baseline characteristics of the patients, with the exception of disease type. The operation time in RvNOTES-H 84.00 (75.00, 95.50) min and SP-LVH 88.00 (73.75, 113.25) min was significantly shorter than RTSP-LH 135.00 (112.25, 154.00) min, (χ² = 60.055, <i>P</i> &lt; 0.001). Compared with SP-LVH and RTSP-LH, RvNOTES-H was superior in lengths of hospital stay, postoperative intestinal function recovery time, and VAS pain scores. There was no significant difference in postoperative complication rates among the three groups. The hospitalization expenses of RTSP-LH and RvNOTES-H were significantly higher than those of SP-LVH.</p> Conclusion <p>The three different single-port surgical approaches were all effective and safe. Compared with SP-LVH and RTSP-LH, RvNOTES-H can reduce postoperative pain score and duration of postoperative exhaust time and hospital stay. While RvNOTES-H offers clinical benefits, its higher cost must be considered in the context of value-based healthcare.</p>