The efficacy of a two-step, oblique incision procedure during 25-gauge vitrectomy on postoperative hypotony was evaluated by a retrospective, case–control study. The transconjunctival incision during 25-gauge vitrectomy was made in two steps: penetration with a microvitreoretinal blade followed by a penetrater instrument of a blunt trocar. The two-step procedure was performed on 89 eyes and with the conventional incision on 68 eyes. The incidence of hypotony (intraocular pressure <6 mmHg) on the first postoperative day and after 1 week and 1 month was compared. Hypotony was found in two eyes (2%) with the two-step method and 12 eyes (18%) with the conventional incision on the first postoperative day (P = 0.001, Fisher's exact probability test). The preoperative intraocular pressure was not significantly different in the two groups but was significantly higher in the two-step group than in the conventional method group on the first postoperative day (P = 0.001, Wilcoxon rank test). Twenty-five-gauge vitrectomy with two-step oblique incisions will reduce the incidence of postoperative hypotony on the first postoperative day.