Eph receptor tyrosine kinases have a wide range of biological functions and have gradually been recognized increasingly as key regulators of inflammation and injury diseases. Although previous studies suggested that EphA2 receptor may be involved in the regulation of inflammation and vascular permeability in injured lung, the detailed effects of EphA2 on LPS-induced acute lung injury (ALI) are still inadequate and the underlying mechanism remains poorly understood. In this study, we detected the effects of EphA2 antagonism on inflammation, pulmonary vascular permeability and oxidative stress in LPS-induced ALI and investigate the potential mechanism. Our results showed that EphA2 antagonism markedly inhibited the cytokines release and inflammatory cells infiltration in BALF, prevented the LPS-induced elevations of MPO activity and MDA level in lung tissues. Our study also found that EphA2 antagonism significantly decreased the wet/dry ratios, reduced the Evans blue albumin extravasation in lung tissues and obviously alleviated the LPS-induced increment of pulmonary vascular permeability. Mechanistically, EphA2 antagonism significantly increased the activation of Nrf2 along with its target antioxidant enzyme HO-1 and inhibited the expressions of TLR4/MyD88 in lung tissues and A549 alveolar epithelial cells. Furthermore, EphA2 antagonism dramatically inhibited the LPS-evoked activations of RhoA/ROCK in lung tissues. In conclusion, our data indicate that EphA2 receptor plays an essential role in LPS-induced ALI and EphA2 antagonism has protective effects against LPS-induced ALI via Nrf2/HO-1, TLR4/MyD88 and RhoA/ROCK pathways. These results suggest that antagonism of EphA2 may be an effective therapeutic strategy for the treatment of ALI. © 2019 Elsevier B.V.