AIM: To improve the standard three-port vitrectomy for establishing and evaluating an endotamponade model in rabbits. METHODS: Three ports were prepared near the third eyelid of rabbits, and the infusion port was placed at the inferior nasal quadrant with the inserted cannula linking with a self-designed handheld rigid infusion catheter. All right eyes of rabbits underwent a modified 25-gauge vitrectomy and were subsequently filled with balanced salt solution, silicone oil, and eight-arm polyethylene glycols (8-arm PEGs) hydrogel separately for comparison. Ophthalmic examinations were performed regularly to record the changes after the surgery. RESULTS: Successful vitrectomy was achieved among 44 chinchilla rabbits. The mean operation time was 4.51±1.25min. Four eyes (9.1%) presented limited lens touch and two eyes (4.5%) showed retinal touch during surgery. Incision leakage was found in three eyes (6.8%) after surgery. There was no endophthalmitis, hemorrhage, or retinal detachment during the observation period and ophthalmic examinations after the implantation of vitreous substitutes. CONCLUSION: The modified technique of the standard vitrectomy applied in the endotamponade model in rabbits shows excellent safety and practicality.