Proliferative vitreoretinopathy (PVR) is a severe complication after rhegmatogenous retinal detachment (RD) surgery and needs further surgical treatments. So far there are no effective drugs for prevention and treatment of PVR. Both integrins αvβ3 and α5β1 are related to human PVR formation. Disintegrins are antagonists of integrins. The drug, a mutant of snake venom-derived disintegrin, also called disintegrin Rhodostomin (Rho) mutant, is a potent antagonist of both integrins αvβ3 and α5β1, of which the effect on PVR remains poorly understood. In vitro assays were used to assess the effects of disintegrin Rho mutant treatment on the proliferation, migration, and adhesion of the retinal pigment epithelial (RPE) cell line derived from human, ARPE-19. In vivo, mice with PVR induction were treated with disintegrin Rho mutant and monitored for PVR severity. In vitro results showed that disintegrin Rho mutant reduced the migration, proliferation, and adhesion of ARPE-19 cells. In vivo results revealed that it could significantly ameliorate the severity of PVR and suppress expression of both integrin αvβ3 and α5β1 and epithelial-mesenchymal transition (EMT) in mouse eyes. Besides, we found decreases in Akt, STAT3, and ERK activation by disintegrin Rho mutant both in vitro and in vivo. In conclusion, disintegrin Rho mutant mitigates experimental PVR with reduced expression of both integrin αvβ3 and α5β1 and presents a potential therapeutic option for PVR in humans.