Objective To observe the effect of epithelial-mesenchymal transdifferentiation (EMT) of human retinal pigment epithelial (RPE) cells induced by vitreous humor in vitro. Methods The third to fifth passage cultured RPE cells were divided into two groups of treatment by 10% serum containing Dulbecco minimum essential medium (DMEM)/F12 medium (group A), or the same medium supplemented with 25% human vitreous (group B). The morphological changes were observed with a phase contrast microscrope. Cell migration, invasion and contractility were tested using a scratch wound assay, Transwell invasion assay and collagen gel contraction analysis. The expression levels of alpha;-smooth muscle actin (SMA) and Snail1 were detected by reverse transcriptase polymerase chain reaction (RT-PCR). Results The RPE cells in group A were flat and gathered together. The RPE cells in group B grew as a fan-shaped projection at one edge and cone-shaped tail at the opposite edge, or spindle-shaped, and appeared to separate. In group A, filamentous actin distributed mainly at the margin of the cells with the distribution an oval shape. In group B, filamentous actin reorganized and formed fan-like flat pseudopodia at one edge of the cells. Compared to group A, the migration and invasion of the cells increased significantly (t=14.190, 22.630; P＜0.05), but contractility decreased remarkably (t=6.221, P＜0.05) in group B. Compared to group A, the expression level of Snail1 mRNA increased significantly (t=3.218, P=0.032), but the expression level of alpha;-SMA mRNA decreased (t=3.990, P=0.016). Conclusions Vitreous humor can induce the EMT of RPE cells. Increasing cell migration, cell invasion, and expression of Snail1 mRNA as well as up-regulated cellsprime; contractility and expression of alpha;-SMA mRNA may be the mechanism.