Background: Acute kidney injury (AKI) is a highly prevalent clinical disorder with significant mortality and no current treatment. The Burns Lab has previously shown that endothelial colony forming cells (ECFCs) release exosomes highly enriched in pro-survival micro-RNA-486-5p. In our mouse model of AKI, intravenous (i.v.) injection of ECFCs or their exosomes protects against kidney ischemic injury, associated with reduction in PTEN, a target of miR-486-5p. Mechanisms mediating recruitment and retention of exosomes are unclear. The interaction of CXC chemokine receptor type 4 (CXCR4) with stromal cell-derived factor (SDF)-1α promotes ECFC adhesion and migration in hypoxic endothelial cells. Whether exosomal miR-486-5p is critical to the prevention of ischemic injury is unclear. The current study aimed to investigate biodistribution and targeting mechanisms of ECFC-derived exosomes, to investigate the delivery and therapeutic potential of miR-486-5p alone, and to determine whether sex differences alter the treatment efficacy. Methods: ECFC-derived exosomes were isolated from cultured media by differential centrifugation and characterized using nanoparticle tracking analysis and immunoblot. Kidney ischemic injury was induced in male and female FVB mice by bilateral renal vascular clamping (30 min). Exosomes (20 µg) or Invivofectamine-mimic complex containing miR-486-5p (1mg/kg) were injected at the start of kidney reperfusion via tail vein. Organs were removed and assays were performed to identify structure and function. In vitro cell studies were also used when necessary. Results: ECFC-derived exosomes preferentially target the ischemic kidney, its endothelium and tubular epithelium, which correlates with increases in miR-486-5p. The transfer of exosomes may be mediated by macropinocytosis by target cells. The SDF-1α/CXCR4 axis plays a role in targeting exosomes to the site of injury. miR-486-5p alone has a similar therapeutic efficacy in preventing ischemia/reperfusion injury as ECFC-exosomes in the mouse model of AKI. Both male and female mice respond to both therapies, however female mice are protected against ischemia reperfusion injury. Conclusions: These results suggest that the protective effects of ECFCs or their exosomes in ischemic AKI may be largely mediated by pro-survival miR-486-5p. These data provide further support for the promising therapeutic potential of ECFC-derived exosomes and miR-486-5p in human AKI.