Significance: Under homeostatic conditions, the endothelium dynamically regulates vascular barrier function, coagulation pathways, leukocyte adhesion, and vasomotor tone. During sepsis and acute inflammation, endothelial cells (ECs) undergo multiple phenotypic and functional modifications that are initially adaptive but eventually become harmful, leading to microvascular dysfunction and multiorgan failure. Critical Issues and Recent Advances: Sepsis unbalances the redox homeostasis toward a pro-oxidant state, characterized by an excess production of reactive oxygen species and reactive nitrogen species, mitochondrial dysfunction, and a breakdown of antioxidant systems. In return, oxidative stress (OS) alters multiple EC functions and promotes a proinflammatory, procoagulant, and proadhesive phenotype. The OS also induces glycocalyx deterioration, cell death, increased permeability, and impaired vasoreactivity. Thus, during sepsis, the ECs are both a significant source and one of the main targets of OS. Future Directions: This review aims at covering the current understanding of the role of OS in the endothelial adaptive or maladaptive multifaceted response to sepsis and to outline the therapeutic potential and issues of targeting OS and endothelial dysfunction during sepsis and septic shock. One of the many challenges in the management of sepsis is now based on the detection and correction of these anomalies of endothelial function.