MicroRNA-494 mediates apoptosis and necrosis in several types of cells, but its renal target and potential role in AKI are unknown. Here, we found that microRNA-494 binds to the 3'UTR of activating transcription factor 3 (ATF3) and decreases its transcription. In mice, overexpression of microRNA-494 significantly attenuated the level of ATF3 and induced inflammatory mediators, such as IL-6, monocyte chemotactic protein-1, and P-selectin, after renal ischemia/reperfusion, exacerbating apoptosis and further decreasing renal function. Activation of NF-κB mediated this proinflammatory response. In this ischemia/reperfusion model, urinary levels of microRNA-494 increased significantly before the rise in serum creatinine. In humans, urinary microRNA-494 levels were 60-fold higher in patients with AKI than normal controls. In conclusion, upregulation of microRNA-494 contributes to inflammatory or adhesion molecule-induced kidney injury after ischemia/reperfusion by inhibiting expression of ATF3. Furthermore, microRNA-494 may be a specific and noninvasive biomarker for AKI.