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To evaluate use of a knotless suture for laparoscopic closure of the equine nephrosplenic space.Experimental in vivo study.Normal horses without previous history of abdominal surgery (n=8).The nephrosplenic space was closed under laparoscopic visualization using a unidirectional, barbed 0 metric absorbable suture (copolymer of glycolic acid and trimethylene carbonate). Intracorporeal suturing of the nephrosplenic space was performed in a cranial-to-caudal direction in a simple continuous fashion. Repeat evaluation was performed laparoscopically in 2 horses and by necropsy in 6 horses. The length of closure was measured and nature of the healed tissue was evaluate grossly.Total surgery time was 65–167 minutes (mean ± SD, 89.6 ± 22.6). Suturing time was 30–65 minutes (40.4 ± 16.3). Second laparoscopy in 2 horses was performed at days 198 and 227. Necropsy was performed at day 69–229 postoperatively (132.7 ± 63.0) in 6 horses. The closure measured 12–14 cm in length (13 ± 1) and consisted of mature fibrous tissue bridging the splenic capsule and the nephrosplenic ligament. No residual suture material was identified grossly in any horses. The procedure was easily performed; extracorporeal suture management to hold it taut was unnecessary since the barbs had excellent purchase in the apposed tissues, and intracorporeal knot tying was not required.The barbed knotless suture appears to be a valid alternative to facilitate laparoscopic closure of the nephrosplenic space in normal horses; however, further work is necessary to investigate its suitability in clinically affected horses.