Acute respiratory distress syndrome (ARDS) is a devastating critical illness disproportionately affecting the elderly population(higher incidence and mortality). The integrity of the lung endothelial cell (EC) monolayer is critical for preservation of lung function. However, mechanisms mediating EC barrier regulation in aging remain unclear. We assessed the severity of acute lung injury (ALI) in young (2 months) and aged (18 months) mice using a two-hit pre-clinical model. Compared to young cohorts, aged mice exhibited increased ALI severity, with greater vascular permeability characterized by elevated albumin influx, levels of bronchoalveolar lavage (BAL) cells (neutrophils) and protein. Aged/injured mice also demonstrated elevated levels of reactive oxygen species (ROS) in the BAL, associated with upregulation of the ROS-generating enzyme, Nox4. We evaluated the role of aging in human lung EC barrier regulation utilizing a cellular model of replicative senescence. Senescent EC populations were defined by increases in beta-galactosidase activity and p16 levels. In response to lipopolysaccharide (LPS) challenge, senescent ECs demonstrate exacerbated permeability responses compared to control "young" ECs. LPS challenge led to a rapid induction of Nox4 expression in both control and senescent ECs, which was post-translationally mediated via the proteasome/ubiquitin system. However, senescent ECs demonstrated deficient Nox4 ubiquitination, resulting in sustained expression of Nox4and alterations in cellular redox homeostasis. Pharmacologic inhibition of Nox4 in senescent ECs reduced LPS-induced alterations in permeability. These studies provide insight into the roles of Nox4/senescence in EC barrier responses and offer a mechanistic link to the increased incidence and mortality of ARDS associated with aging.