BACKGROUND AND PURPOSE: Ischemic stroke is a major cause of death worldwide but lacks viable treatment or treatment targets. Monocyte locomotion inhibitory factor (MLIF) is a small heat-stable pentapeptide produced by Entamoeba histolytica in axenic culture, which is supposed to protect the brain from ischemic injury; the mechanism, however, remains unknown. In this study, we further investigated the mechanism underlying the protective role of MLIF in brain ischemia. METHODS: A middle cerebral artery occlusion model in rats was used for detecting the effect of MLIF in the brain ischemia in vivo. To identify targets of MLIF in brain endothelial cells, we performed immunoprecipitation of biotin-conjugated MLIF and mass spectrometry. RESULTS: MLIF can protect the brain from ischemic injury in vivo, yielding decreased ischemic volume, prolonged survival, and improved neurological outcome. In vitro studies showed that MLIF displayed protective effects through inhibition of expression of pathological inflammatory adhesion molecules and enhancing endothelial nitric oxide synthase expression and nitric oxide release in the cerebrovascular endothelium. The target screening experiments demonstrated binding of MLIF to the ribosomal protein translation elongation factor eEF1A1. MLIF enhanced endothelial nitric oxide synthase expression through stabilization of endothelial nitric oxide synthase mRNA, and eEF1A1 was shown to be necessary for this enhanced expression. Knockdown of eEF1A1 or inhibition of endothelial nitric oxide synthase attenuated MLIF-mediated inhibition of adhesion molecule expression. CONCLUSIONS: In this study, we identified a new potential pharmacologically targetable mechanism underlying MLIF's protective effects in brain ischemia through the eEF1A1/endothelial nitric oxide synthase pathway.