OBJECTIVE: To explore whether two-layer laparoscopic vaginal cuff closure at the time of laparoscopic hysterectomy is associated with a lower rate of postoperative complications compared with a standard one-layer cuff closure. METHODS: A retrospective cohort study of total laparoscopic hysterectomies performed by fellowship-trained minimally invasive gynecologic surgeons between 2011 and 2017 was performed. Surgeons sutured the vaginal cuff laparoscopically, either in a two- or one-layer closure. The primary outcome was a composite of total postoperative complications, including all medical and surgical complications within 30 days and vaginal cuff complications within 180 days. Factors known to influence laparoscopic vaginal cuff complications including age, postmenopausal status, body mass index, tobacco use, and immunosuppressant medications were examined and controlled for, while surgeon skill, colpotomy technique, and suture material remained standardized. We conducted statistical analyses including χ2, Fisher exact test, logistic regression, and post hoc power calculations. RESULTS: Of the 2,973 women who underwent total laparoscopic hysterectomies, 40.8% (n=1,213) of vaginal cuffs were closed with a two-layer closure and 59.2% (n=1,760) with a one-layer technique. Two-layer vaginal cuff closure was associated with decreased numbers of total postoperative complications (3.5% vs 5.7%; P<.01). The primary difference stemmed from lower vaginal cuff complications within 180 days (0.9% vs 2.6%; P<.01); no differences in 30-day medical and surgical postoperative complications were observed between the two groups (2.6% vs 3.1%; P=.77). No patients in the two-layer vaginal cuff closure cohort experienced a vaginal cuff dehiscence or mucosal separation compared with 1.0% in the one-layer group (P<.01). Compared with a one-layer closure, a two-layer closure was protective from postoperative complications (adjusted odds ratio 0.38, 95% CI 0.19–0.74). CONCLUSION: Although postoperative complications with laparoscopic hysterectomies are rare, two-layer laparoscopic vaginal cuff closure is associated with lower total postoperative complications compared with a one-layer closure. The difference was primary driven by cuff complications.