Renal tubular epithelial cell (RTEC) death and renal interstitial inflammation are the most crucial pathophysiological changes in acute kidney ischemia/reperfusion injury (IRI). The microRNA (miR)-181d family plays diverse roles in cell proliferation, apoptosis and inflammation, but its renal target and potential role in IRI are unknown. Here, we showed that the expression of miR-181d-5p decreased and Krueppel-like factor 6 (KLF6) increased in a renal cell (HK-2) model of hypoxia/reoxygenation (H/R) injury and a mouse model of renal IRI. They were mainly distributed in the renal tubules. After renal IRI, miR-181d-5p overexpression significantly inhibited inflammatory mediators, reduced apoptosis and further improved renal function. KLF6 exacerbated RTEC damage and acted as a NF-κB co-activator to aggravate the renal IRI inflammatory response. Mechanistically, KLF6 was predicted as a new potential target gene of miR-181d-5p through bioinformatic analysis and luciferase reporter assay verification. After overexpressing miR-181d-5p and inhibiting KLF6, the role of miR-181d-5p was weakened on the renal damage improvement. In conclusion, miR-181d-5p upregulation produced protective antiapoptotic and anti-inflammatory effects against IRI in kidneys and H/R injury in HK-2 cells , and these effects were achieved by targeted inhibition of KLF6. Thus, our results provide novel insights into the molecular mechanisms associated with IRI and a potential novel therapeutic target.