The development of a 25-gauge vitrectomy system represents a major step forward in vitreoretinal surgery. The main advantages of small-gauge incisions are reduction of surgical time, decreased postoperative inflammation, and fast postoperative recovery time. The use of this technique requires research, training, knowledge, and experience on the part of the vitreoretinal surgeon and the personnel in the operation room. At the same time the indications for 25-gauge vitrectomy have changed a lot in the last years due to improvement in materials and technology and the advent of new instruments. Nowadays, with the development of new surgical instrumentation and the improvement of surgical skills, we can approach the whole spectrum of vitreoretinal pathology. This chapter describes the instruments used in 25-gauge vitrectomy, explains how to set the vitrectomy machine for safe surgery and what the indications for 25-gauge vitrectomy are (with particular reference to vitreoretinal interface diseases, retinal detachment, and proliferative diabetic retinopathy) as well as the complications that can occur, and provides some pearls for challenging cases.