To investigate the risk of vaginal cuff dehiscence after different routes of hysterectomy and methods of cuff closure. A multi-institutional analysis of 12,398 patients who underwent hysterectomy for both benign and malignant disease between 1994 and 2008 was performed. We analyzed how different routes of hysterectomy and approaches to cuff suture may influence the risk of development of vaginal dehiscence. Women who had total laparoscopic (n=3,573), abdominal (n=4,291), and vaginal (n=4,534) hysterectomies experienced 23 (0.64%), 9 (0.2%), and 6 (0.13%) cases of vaginal cuff dehiscence, respectively. Total laparoscopic hysterectomy was associated with a higher incidence of cuff separations, compared with abdominal hysterectomy (0.64% compared with 0.21%, P=.003) and vaginal hysterectomy (0.64% compared with 0.13%, P<.001). Within the endoscopic group, patients who underwent vaginal closure with laparoscopic knots had a higher rate of cuff dehiscence than patients who had suture with transvaginal knots (20 of 2,332 [0.86%] compared with 3 of 1,241 [0.24%], P=.028). When vaginal suture was performed transvaginally, no statistical difference in vaginal cuff dehiscence rate was observed compared with both abdominal hysterectomy (0.24% compared with 0.21%, P=.83) and vaginal hysterectomy (0.24% compared with 0.13%, P=.38). Use of monopolar energy at the time of colpotomy and reducing the power of monopolar energy from 60 watts to 50 watts when colpotomy was performed at the end of total laparoscopic hysterectomy did not alter the rate of cuff separations. Transvaginal suturing appears to reduce the risk of vaginal dehiscence after total laparoscopic hysterectomy. II