Objective: To investigate the long-term outcomes and optimal timing of surgery for Mayer–Rokitansky–Küster–Hauser syndrome (MRKH) patients undergoing vaginoplasty using an acellular porcine small intestinal submucosa graft (SIS). Design: Observational study. Setting and Population: Seventy-eight MRKH syndrome patients, forty-eight healthy controls and a post-SIS patient who delivered a baby following the world’s first robot-assisted uterus transplantation. Methods: The outcome of sexual function and psychological were assessed through three standard questionnaires: the female sexual function index (FSFI), Self-rating scale of body image (SSBI) and self-acceptance questionnaire (SAQ). Main Outcome Measures: Sexual function and psychology outcomes. Results: A total of 89.7% patients achieved a “good” vaginal width, and the mean vaginal length was 6.92±0.80 cm. The overall FSFI score of MRKH syndrome patients was significantly lower than that of healthy controls (24.43±4.42 versus 30.28±2.61, P<0.001). Three factors (sexual arousal, orgasm and sexual satisfaction score) in the FSFI increased significantly as the postoperative time increased. The emotional impairment factor of the SSBI showed a higher score in MRKH syndrome patients (1.56±1.83 versus 1.15±2.05, P=0.030). The psychological scale score showed an improving trend as the postoperative time was prolonged in MRKH syndrome patients. Conclusions: SIS surgery is effective in improving the patient’s sexual function and psychological problems, and this effect is more pronounced in patients who have the surgery earlier. It is recommended to undergo surgery when the patient is physiologically mature and expresses the desire to proceed with therapy.