Pulmonary fibrosis, a secondary effect of other diseases such as sepsis-associated ARDS, interferes with a patient's ability to breathe. The control of proliferation and differentiation become a strategy for treating pulmonary fibrosis. In this study, we want to explore the effects of Lipopolysaccharide (LPS) on the activity of RhoA/ROCK1 signaling pathways, and tried to explore the mechanism of the signaling pathways in MRC-5 cells proliferation and differentiation. We divided cells into four groups, control group, LPS group, LPS + low-dose Fasudil (15 mu mol/ml) group and LPS + high-dose Fasudil (30 mu mol/ml) group. RhoA activity was determined by Rho pull-down analysis and the protein levels of GTP-RhoA, ROCK1, MYPT-1 (Myosin phosphatase target subunit), p-MYPT-1 (a downstream substrate of ROCK1) and alpha-smooth muscle (alpha-SMA, a marker of myofibroblast cells differentiation) were determined by Western blot. Real-time PCR was used to determine the level of alpha-SMA mRNA. Cell proliferation rate was examined using CCK-8 and EdU Imaging Kit. LPS up-regulated RhoA activity, protein expressions of ROCK1, p-MYPT-1 and alpha-SMA as well as proliferation rate (P<0.05). Furthermore, the effects mentioned above were inhibited by Fasudil (a highly selective inhibitor of ROCK) in dose-dependent manner. The significant higher inhibitory effects in high-dose Fasudil group were observed compared with low-dose Fasudil. Our data suggest that LPS induced MRC-5 cells proliferation and differentiation via RhoA/ROCK1 signaling pathways. Fasudil attenuated LPS-induced cells proliferation and differentiation by inhibiting this signaling pathway. Regulating RhoA/ROCK1 signaling pathway could be a potential new target to treat pulmonary fibrosis.