AIM: To investigate the regulatory mechanism of glycogen synthase kinase 3β (GSK3β) in epithelial-mesenchymal transition (EMT) process after proliferative vitreoretinopathy (PVR) induction. METHODS: Experimental PVR was induced by intravitreal injection of retinal pigment epithelium (RPE) cells in the eyes of rabbits. A PI3K/Akt inhibitor (wortmannin) and a GSK3β inhibitor (LiCl) were also injected at different time during PVR progress. Electroretinogram (ERG), ocular fundus photographs, and B-scan ultrasonography were used to observe the PVR progress. Western blot test on the extracted retina were performed at 1, 2, 4wk. The expression of the mesenchymal marker vimentin was determined by immunohistochemistry. Toxicity of wortmannin and LiCl were evaluated by ERG and TdT-mediated dUTP nick-end labeling (TUNEL) assay. The vitreous was also collected for metabolomic analysis. RESULTS: Experimental PVR could significantly lead to EMT, along with the suppressed expression of GSK3β and the activation of Wnt/β-catenin and PI3K/Akt pathways. It was verified that upregulating the expression of GSK3β could effectively inhibit EMT process by suppressing Wnt/β-catenin and PI3K/Akt pathways. CONCLUSION: GSK3β effectively inhibits EMT via the Wnt/β-catenin and PI3K/Akt pathways. GSK3β may be regarded as a promising target of experimental PVR inhibition.