Objective:To explore the application of real-time transrectal ultrasound (TRUS) during seminal vesiculoscopy in infertile men with azoospermia or oligoasthenospermia.Methods:We retrospectively analyzed the clinical data of 25 cases of azoospermia or oligoasthenospermia due to ejaculate ducts obstruction who were treated with real-time transrectal ultrasound-guided seminal vesiculoscopy between September 2011 and December 2015. Patients’ age was(29.4±4.5) years. All patients accepted semen analysis, serum sex hormone, MRI, TRUS and then diagnosed as obstructive azoospermia, and 13 cases had intractable obstructive azoospermia or oligoasthenospermia after the failure of simple seminal vesiculoscopy(the path to the ejaculatory duct and seminal vesicle couldn’t be found). All patients were treated with seminal vesiculoscopy under real-time guidance with TRUS. We assessed the success rate of surgery, surgical time and complications.Results:The scope was successfully inserted into the seminal vesicle in 21 of the 25 cases (success rate, 84%). The median operative time was 75(31, 148) min. None of the patients developed severe complications. Among 4 failure cases (4/25, 16%), 1 was due to abnormal congenital development. In 2 cases, a clear outlet of the dual ejaculatory duct could not be found after it was inserted into the prostatic utricle. One case was considered as a Müllerian tubular cyst, and the seminal vesicle scope was used to assess the cystic side wall. The 21 patients were followed up for 3 to 6 months, semen volume 2.0(0-5.2)ml, total sperm 28(0-832) ×10 6/ejaculate, sperm density 5.6(0-110.3)×10 6/ml, mobility rate of sperm 5.4%(0-63.6%), and the differences were significant as compared to that before the surgery [semen volume 0.4(0-2.8)ml, total sperm 0(0-342)×10 6/ejaculate, sperm density 0(0-90.7)×10 6/ml, mobility rate of sperm 0(0-24.1%), all P<0.05]. Among the 17 patients who underwent follow-up of 5 to 9 years, 3 patients was conceived naturally and 9 patients’ postoperative sperm quality has improved and pregnancy in vitro fertilization by extracting sperm from semen. Conclusions:Intraoperative real-time transrectal ultrasound guidance can improved the success rate of seminal vesiculoscopy and promoted operative safety.