Introduction and objective Laparoscopic suturing of the vaginal cuff and knotting is the most challenging step in total laparoscopic hysterectomy (TLH) and requires surgical skill. The objective of this study was to compare the efficacy and safety of unidirectional barbed suture (V-LocTM 180; Covidien, Mansfield, MA) with the conventional polyglactin 910 suture (coated Vicryl; Covidien) for vaginal cuff closure in patients with benign uterine diseases undergoing total laparoscopic hysterectomy. Methods  A prospective observational study was carried out at the department of obstetrics and gynecology, All India Institute of Medical Sciences (AIIMS), Rishikesh, for two years. A total of 109 patients with benign uterine diseases planned for TLH were included in the study. Laparoscopic vaginal cuff closure was performed with the unidirectional barbed suture (V-Loc) in 44 patients and with the standard polyglactin 910 suture (Vicryl) in 65 patients. The primary outcome measure was vaginal cuff closure time. Secondary outcome measures included total operative time, blood loss, average number of stitches, postoperative pain perception, duration of hospital stay, vaginal cuff-related complications, and dyspareunia. Results Demographic variables and baseline characteristics were similar in both groups except for body mass index (BMI). The mean vaginal cuff closure time was significantly less in the V-Loc group (8.84 ± 2.18 min) than in the Vicryl group (11.66 ± 1.74 min) (p = <0.01). Mean operative time was comparable in both groups (V-Loc group - 109.36±33.02 and Vicryl group - 108.49±40.48; p = 0.91). Other intraoperative parameters, such as blood loss and number of stitches in cuff closure, and postoperative characteristics, such as pain score, duration of hospital stay, vaginal cuff-related complications (vault cuff dehiscence, hematoma, or abscess), and dyspareunia, were comparable in both the groups. Conclusions The unidirectional barbed suture significantly reduces vaginal cuff closure time. It is a safe, effective, and well-tolerated alternative to conventional Vicryl suture for vaginal cuff closure in TLH without increasing the risk of postoperative vaginal complications particularly where affordability is not an issue and resources are accessible.