Disruption of endothelial cell (ECs) and pericytes interactions results in vascular leakage in acute lung injury (ALI). However, molecular signals mediating EC–pericyte crosstalk have not been systemically investigated, and whether targeting such crosstalk could be adopted to combat ALI remains elusive. Using comparative genome-wide EC–pericyte crosstalk analysis of healthy and LPS-challenged lungs, we discovered that crosstalk between endothelial nitric oxide and pericyte soluble guanylate cyclase (NO–sGC) is impaired in ALI. Indeed, stimulating the NO–sGC pathway promotes vascular integrity and reduces lung edema and inflammation-induced lung injury, while pericyte-specific sGC knockout abolishes this protective effect. Mechanistically, sGC activation suppresses cytoskeleton rearrangement in pericytes through inhibiting VASP-dependent F-actin formation and MRTFA/SRF-dependent de novo synthesis of genes associated with cytoskeleton rearrangement, thereby leading to the stabilization of EC–pericyte interactions. Collectively, our data demonstrate that impaired NO–sGC crosstalk in the vascular niche results in elevated vascular permeability, and pharmacological activation of this crosstalk represents a promising translational therapy for ALI.