Exposure to hyperoxia results in acute lung injury. A pathogenic consequence of hyperoxia is endothelial injury. Macrophage migration inhibitory factor (MIF) has a cytoprotective effect on lung endothelial cells; however, the mechanism is uncertain. We postulate that the MIF receptor CD74 mediates this protective effect. Using adult wild-type (WT), MIF-deficient (Mif−/−), CD74-deficient (Cd74−/−) mice and MIF receptor inhibitor treated mice, we report that MIF deficiency or inhibition of MIF receptor binding results in increased sensitivity to hyperoxia. Mif−/− and Cd74−/− mice demonstrated decreased median survival following hyperoxia compared to WT mice. Mif−/− mice demonstrated an increase in bronchoalveolar protein (48%) and lactate dehydrogenase (LDH)(68%) following 72 hours of hyperoxia. Similarly, treatment with MIF receptor antagonist resulted in a 59% and 91% increase in bronchoalveolar lavage protein and LDH, respectively. Inhibition of CD74 in primary murine lung endothelial cells (MLECs) abrogated the protective effect of MIF, including decreased hyperoxia-mediated AKT phosphorylation and a 20% reduction in the antiapoptotic effect of exogenous MIF. Treatment with MIF decreased hyperoxia-mediated H2AX phosphorylation in a CD74-dependent manner. These data suggest that therapeutic manipulation of the MIF-CD74 axis in lung endothelial cells may be a novel approach to protect against acute oxidative stress.—Sauler, M., Zhang, Y., Min, J.-N., Leng, L., Shan, P., Roberts, S., Jorgensen, WL, Bucala, R., Lee, PJ Endothelial CD74 mediates macrophage migration inhibitory factor protection in hyperoxic lung injury.