Renal ischemia-reperfusion injury (IRI) is a common complication in several clinical scenarios including kidney transplantation. Mannan-binding lectin-associated serine proteinase (MASP)-2 is essential for activation of the complement lectin pathway, which has been implicated in the pathogenesis of renal IRI and therefore represents a potential therapeutic target. We developed a new, affinity-enhanced MASP-2 inhibitor, EVO24, by directed evolution of the D2 domain of human tissue factor pathway inhibitor. EVO24 was fused with a human IgG1-Fc to create the homodimer EVO24L, which potently and selectively inhibited the lectin pathway in human and mouse serum in vitro. EVO24L was tested in a mouse model of unilateral warm renal IRI. EVO24L administered before and after ischemia significantly protected against IRI, with improved renal function as well as reduced tubular injury and inflammatory cell infiltration at 24 h compared to vehicle-treated mice. Immunofluorescence analyses showed reduced deposition of complement components (C3d, C4d, and C9) and reduced expression of VCAM-1, indicating a decrease in complement activation and endothelial cell activation. Additionally, EVO24L treatment lowered plasma levels of complement C5a, hyaluronan (a marker of endothelial glycocalyx shedding), and the proinflammatory cytokines IL-6 and TNF-α. Our findings indicate that EVO24L inhibits acute inflammatory responses in renal IRI by blocking the lectin pathway, confirming the important role of this pathway in acute ischemic kidney injury and warranting further investigation of EVO24L in clinical settings.