Background: Pulmonary microvascular endothelial cells (PMVECs) are initial targets of sepsis-induced acute lung injury (ALI). During the apoptosis of PMVECs, tight junctions (TJ) and adherens junctions (AJ) are firstly damaged. Previous studies have suggested hydrogen treatment can protect lung microvasculature of mice from sepsis-induced endothelial dysfunction and maintain the coherence of pulmonary endothelium, but the underlying mechanism remains unclear. Methods: We investigated the role of hydrogen-rich medium on regulating intercellular junction proteins under lipopolysaccharide (LPS) treatment which mimicked sepsis in vitro. Changes of cytoskeleton regulatory protein ROCK and RhoA as well as PMVEC apoptotic rate were examined. Results: LPS treatment reduced the expression levels of occludin and VE-cadherin in PMVECs, while hydrogen-rich medium can recover these changes. Furthermore, H2 can significantly ameliorate the excessive expression of ROCK and RhoA under sepsis-mimicking condition. The application of RhoA activator U-46619 resulted in a more significant elevation in cell apoptotic rate as well as reduction in the expression of junctional proteins. Using H2 can almost completely inhibit the effects of RhoA activator. Conclusions: Our findings suggest that RhoA is a crucial protein in the signaling pathway of LPS-induced endothelial cell dysfunction. Hydrogen treatment can prevent LPS-induced junctional injury and cell death by inhibiting the activity of RhoA.