Background: This study evaluates the clinical effects of the stepwise anterior vitrectomy on the prevention of positive vitreous pressure (PVP) during penetrating keratoplasty (PKP). Methods: PKP in conjunction with stepwise anterior vitrectomy was performed on 15 eyes of 15 patients under retrobulbar anesthesia. A preset vitrectomy trocar-cannula was inserted into the vitreous cavity before PKP. During the opening of the anterior chamber, intermittent vitrectomy and corneal incision expansion were performed alternately to keep the lens or artificial intraocular lens (IOL) and iris flat until the entirety of the pathological cornea had been dissected. The main outcome measures include visual acuity, crystalline lens rise (CLR), corneal curvature and diopter, and corneal endothelial cell loss. Results: All surgical procedures were performed successfully without any PVP-related intraoperative complications. The mean time of the stepwise vitrectomies was 3.1 ± 0.7 s, the duration of each vitrectomy was 8.1 ± 5.3 s, and the duration of the total surgery was 60.5 ± 5.3 min. The anterior segment reaction was mild and the shape of the pupil remained normal 1 day after surgery. The mean preoperative and mean 3-month postoperative CLR values were 0.48 ± 0.09 mm and-0.16 ± 0.04 mm, respectively (p \ 0.0001). The mean preoperative endothelial cell density in donor buttons was 2570 ± 171 cells/mm 2 , and the mean 6-and 12-month postoperative endothelial cell density in donor buttons was 2207 ± 127 cells/mm 2 and 2000 ± 198 cells/ mm 2 , respectively. Conclusions: The novel and stabilized PKP procedure, performed in conjunction with the stepwise anterior vitrectomy, effectively avoided the PVP during open-sky surgery.