Trans-endothelial hyper-permeability caused by numerous agonists is dependent on Heat shock protein 90 (Hsp90) and leads to endothelial barrier dysfunction (EBD). Inhibition of Hsp90 protects and restores trans-endothelial hyper-permeability. Hyper-acetylation of Hsp90, as by inhibitors of histone deacetylase (HDAC), suppresses its chaperone function and mimics the effects of Hsp90 inhibitors. In this study we assessed the role of HDAC in mediating LPS-induced trans-endothelial hyper-permeability and acute lung injury (ALI). We demonstrate that HDAC inhibition protects against LPS-mediated EBD. Inhibition of multiple HDAC by the general inhibitors panobinostat or trichostatin provided protection against LPS-induced trans-endothelial hyper-permeability, acetylated and suppressed Hsp90 chaperone function and attenuated RhoA activity and signaling crucial to endothelial barrier function. Treatment with the HDAC3-selective inhibitor, RGFP966, or the HDAC6-selective inhibitor tubastatin A, provided partial protection against LPS-mediated trans-endothelial hyper-permeability. Similarly, knock down of HDAC3 and HDAC6 by specific siRNAs provided significant protection against LPS-induced EBD. Furthermore, combined pharmacologic inhibition of both HDAC 3 and 6 attenuated the inflammation, capillary permeability and structural abnormalities associated with LPS-induced ALI in mice. Together these data indicate that HDAC mediate increased trans-endothelial hyper-permeability caused by LPS and that inhibition of HDAC protects against LPS-mediated EBD and ALI by suppressing Hsp90-dependent RhoA activity and signaling.