To evaluate the efficiency, preliminary safety, and feasibility of a 27-gauge instrument system for transconjunctival microincision vitrectomy surgery (MIVS) in a variety of vitreoretinal diseases. Experimental, interventional case series. Thirty-one eyes (31 patients) underwent a variety of vitreoretinal procedures using the 27-gauge transconjunctival MIVS system to treat epiretinal membrane (n = 10), idiopathic macular holes (n = 7), diabetic vitreous hemorrhage (n = 5), vitreous opacity with suspicion of intraocular lymphoma (n = 4), focal diabetic traction retinal detachment (n = 3), macular traction syndrome (n = 1), and macular edema secondary to central retinal vein occlusion (n = 1). We developed a 27-gauge instrument system that includes an infusion line, a high-speed vitreous cutter, an illumination system, and a variety of vitreoretinal instruments, such as membrane forceps and sharp-tipped endophotocoagulation probes. The duty cycle of the 27- and 25-gauge cutters was measured for several cut rates using a high-speed imaging camera. Infusion and aspiration rates were measured using balanced saline solution (BSS) and porcine vitreous with different aspiration levels. Surgical outcomes, including anatomic success, visual outcomes, operating times, and intraoperative and postoperative complications, were evaluated. Duty cycle of cutters, infusion and aspiration rates, and surgical results of 27-gauge vitrectomy. Although the infusion and aspiration rates of the 27-gauge system measured in BSS were reduced to an average of 62% and 80%, respectively, compared with those of the 25-gauge system, the duty cycle of the 27-gauge cutter, 61% at 1000 cpm and 38% at 1500 cpm, was equal to or better than those of the 25-gauge cutter (62% and 28%, respectively). Analysis of the fluid dynamics showed that vented gas-forced infusion can be set to range from 20 to 30 mmHg to control intraocular pressure (IOP) during 27-gauge vitrectomy. Anatomic success was achieved in all study eyes (100%); 20 eyes (65%) had visual improvement of 3 lines or more. No eyes required conversion to larger gauge instrument. All sclerotomies self-sealed without hypotony (IOP < or = 7 mmHg) from 1 day postoperatively. Although the fluid dynamics and cutting efficiency of 27-gauge instruments are lower compared with 25-gauge MIVS, the 27-gauge system is feasible and may reduce concerns about wound sealing-related complications in selected cases. Proprietary or commercial disclosure may be found after the references.