Transcriptional repressor activating transcription factor 3 (ATF3) is induced by various stress stimuli, including inflammation‐induced renal injury. In addition, ATF3 also down‐regulates adhesion molecules like intercellular adhesion molecule (ICAM), vascular cell adhesion molecule (VCAM), and monocyte chemotactic protein‐1 (MCP‐1). However, the relation between up‐regulated ATF3 after renal ischemia/reperfusion (I/R) injury and MCP‐1 is not completely understood. In this study, we demonstrated that, in renal I/R induced inflammation, induction of adhesion molecules (interleukin‐6, P‐selectin, E‐selectin, ICAM, VCAM, and MCP‐1) was higher in ATF3‐knockout mice than in wild‐type animals. Molecular and biochemical analyses revealed that ATF3 binds to the ATF/CRE sites in the MCP‐1 promoter and inhibits the secretion of MCP‐1 from renal epithelial cells after I/R injury. Urinary exosome containing ATF3 RNA was 60‐fold higher in patients with acute kidney injury than in normal controls, but no difference in total urinary ATF3 RNA levels was found. In addition, in vitro study showed that exosome containing ATF3 RNA derived from epithelial cells also inhibits MCP‐1 expression in the epithelial cells and macrophage migration. Furthermore, direct administration of the epithelium‐derived exosomal ATF3 RNA attenuates I/R induced kidney injury. Together, our studies reveal a novel regulatory mechanism of MCP‐1 expression mediated by the exosomal ATF3 RNA under renal I/R insult and suggest a potential targeted therapy for I/R induced acute kidney injury. J. Cell. Physiol. 229: 1202–1211, 2014. © 2014 Wiley Periodicals, Inc.