To determine the incidence of endophthalmitis following 25-gauge and standard 20-gauge vitrectomy. In this single-centre retrospective interventional case series, we evaluated the incidence of acute endophthalmitis occurring within the 14-day postoperative period in all patients who underwent pars plana vitrectomy between 1 November 2002 and 31 December 2006. A total of 3477 consecutive patients were identified. Of them, 3046 patients underwent 20-gauge vitrectomy and 431 underwent 25-gauge vitrectomy. The event rates of postoperative endophthalmitis were 0.03% (1 of 3046) after 20-gauge vitrectomy and 0.23% (1 of 431) after 25-gauge vitrectomy (P=0.23). In the endophthalmitis case that occurred after 25-gauge vitrectomy, a combined phacoemulsification cataract surgery was performed with the 25-gauge vitrectomy. Analyses of event rates of postoperative endophthalmitis after combining phacoemulsification cataract surgery and vitrectomy were 0% (0 of 170) for 20-gauge vitrectomy and 2.17% (1 of 46) for 25-gauge surgery vitrectomy (P=0.21). The incidence of postoperative endophthalmitis following 25-gauge vitrectomy and 20-gauge surgery is low. Although there is a trend suggesting an increased risk of endophthalmitis with the 25-gauge system, this difference was not statistically significant given the low number of measured outcomes.