Background: Acute kidney injury (AKI) can result from renal ischemia and reperfusion (I/R) and often occurs during surgical procedures in cardiac, liver, kidney transplantation, and trauma-hemorrhage. Milk fat globule epidermal growth factor-factor VIII (MFG-E8) functions as a bridging molecule to promote the removal of dying cells by professional phagocytes. Because MFG-E8 promotes clearance of apoptotic cells, we have explored its therapeutic potential in various organ injury conditions. To develop human MFG-E8 as a potential therapy, we have generated a human cell-expressed, and thus glycosylated, tag-free recombinant human (rh) MFG-E8 and tested its safety and biological activity in vitro . We hypothesize that the tag-free glycosylated rhMFG-E8 is protective in I/R-induced AKI and it can be developed as an effective therapy for AKI. Methods: To assess the pharmacokinetic properties of the tag-free rhMFG-E8, Sprague-Dawley rats were either untreated or treated with a bolus dose of the tag-free rhMFG-E8, blood collected at various time points and the recovery of human MFG-E8 in the blood were measured by ELISA. Adult male C57BL6 mice underwent bilateral renal ischemia for 30 min, and immediately upon reperfusion, mice were treated intraperitoneally with either normal saline (vehicle) or 20 μg/kg human cell expressed, glycosylated tag-free rhMFG-E8. At either 24 h or 48 h after I/R, blood and kidneys were harvested for further analysis. In separate cohorts of mice after I/R and treatment, mice were observed for 10 days, and survival recorded. Results: AKI rats treated with the tag-free rhMFG-E8 had similar half-life as those in the treated control rats. At 48 h after I/R-induced AKI, renal function markers, blood urea nitrogen, and creatinine were increased and treatment with the tag-free rhMFG-E8 significantly decreased these markers. At both 24 h and 48 h after AKI, inflammatory cytokines, TNF-α, IL-6, and IL-1β were increased and treatment decreased these levels. The kidney mRNA expressions of these cytokines were also increased at 24 h after AKI and treatment significantly decreased those mRNA expressions. Histologically, at 48 h after AKI, tubular damage, and the number of TUNEL staining cells were increased and treatment markedly decreased these measurements. Administration of tag-free rhMFG-E8 at the time of reperfusion improved survival in a 10-day survival study. Conclusion: Our new human cell-expressed tag-free rhMFG-E8 is protective in I/R-induced AKI and it may have the potential to be further developed as a safe and effective therapy for AKI.