Objective To explore the effect and mechanism of Rho GDP dissociation inhibitor α (Rho GDIα) knockdown on epithelial-mesenchymal transition (EMT) in silicosis. Methods The alveolar type Ⅱ epithelial cell line A549 was induced by transforming growth factor-β1(TGF-β1) for EMT.The cells were divided into four groups: lentivirus empty vector (LEV) group, RhoGDIα depletion lentiviral vector (Lv-Rho GDIα-inhibition)group, LEV-transfected cells induced by TGF-β1 (LEV+TGF-β1) group, and Lv-Rho GDIα-inhibition transfected cells induced by TGF-β1 (Lv-Rho GDIα-inhibition+TGF-β1) group. Western blotting and immunocytochemical staining were used to examine the protein expression of Rho GDIα, RhoA, Rho kinase(ROCK), E-cadherin (E-cad), α-smooth muscle actin(α-SMA) and collagen-I. Results Compared with the LEV control group, Rho GDIα, RhoA, ROCK, α-SMA and collagen-I showed upregulated expression, while E-cad showed downregulated expression in the Lv-Rho GDIα-inhibition group. Compared with the LEV + TGF-β1 group, Rho GDIα, RhoA, ROCK, α-SMA and collagen-I were all upregulated, and E-cad was downregulated in the Lv-Rho GDIα-inhibition+TGF-β1 group. Western blotting and Cell Counting Kit-8 result showed that silencing of Rho GDIα inhibited cell proliferation. Conclusions Rho GDIα knockdown may lead to the inhibition of epithelial-mesenchymal transformation by regulating the RhoA/ ROCK signaling pathway, and plays an anti-silicosis effect.