Internal hernia (IH) after Roux-Y gastric bypass (RYGB) can lead to extended small bowel ischemia if it not recognized and treated promptly. The aim of this study is to show whether improvement in mesenteric defect (MD) closure reduces the incidence of IH. Retrospective analysis of prospectively collected data from our database including all patients who underwent laparoscopic RYGB between 1999 and 2015. The usual technique was a retrocolic/retrogastric RYGB. We divided patients in four groups according to the closure technique for MD and compared incidences of IH between groups. All patients had at least 8years of follow-up. A total of 1927 patients (1497 females/460 males, mean age of 41.5 ± 11years) were operated. A retrocolic/retrogastric RYGB was performed in 1747 (90.7%) and an antecolic RYGB in 180 patients. Mean duration of follow-up was 15 (8-24) years. 111 patients (5.8%) developed IH, the majority through the jejunojejunostomy (JJ, 3.7%) and Petersen (1.7%) defects. With improvement of closure technique, the incidence decreased over time, from 12.9% in the group with separate sutures to 1.05% in the most recent group with running non-absorbable sutures and an additional purse-string at the JJ defect (p < 0.0001). Meticulous closure of MD during RYGB is a very important step that significantly reduces the IH risk after RYGB, even with a retrocolic/retrogastric anatomy. Using running non absorbable braided sutures and an additional purse-string suture at the JJ is the most effective technique, but a small IH risk persists. A high index of suspicion remains necessary in patients who present with acute abdominal pain after RYGB.