Recovery from acute kidney injury includes restoration of GFR and tubular repair but may be associated with impaired renal hemodynamic and natriuretic responses that contribute to the development of salt-sensitive hypertension and chronic kidney disease. NFAT5, a transcription factor that protects the kidney from hypertonic stress also is activated by hypoxia, however, a functional role for this transcription factor in IRI has not been established. Mice were subjected to IRI by clamping of the left renal pedicle for 30 min followed by reperfusion for 3 or 48 hr. After 3 hr, analysis by qRT-PCR showed that the relative abundance of NFAT5 mRNA was similar in mTAL tubules, or outer medulla (OM), prepared from contralateral and clamped kidneys. However, after 48 hr NFAT5 mRNA accumulation increased approximately three-fold in both mTAL tubules (NFAT5/β-actin, 3.14 ± 0.32 to 0.95 ± 0.11, p<0.05) and OM (2.71 ± 0.43 to 0.82 ± 0.22, p<0.05). Mice were then either pretreated for 72 hr with an intrarenal injection of a lentivirus shRNA construct to knockdown functional NFAT5 (EGFP-U6-N5-ex8, n=6), or a control vector (EGFP-U6, n=6) before induction of IRI. NGAL and Kim1, established biomarkers of IRI, mRNA was dramatically increased after IRI and was further increased after knockdown of NFAT5 (NGAL: EGFP-U6-N5-ex8; 111.5 ± 10.9, EGFP-U6; 59 ± 6.2, p<0.05 and Kim1: EGFP-U6-N5-ex8; 25.8 ± 3.8, EGFP-U6; 12.9 ± 2.1, p<0.05), suggesting that inhibition of NFAT5 exacerbates damage in the TAL after IRI. Apoptotic cells in OM of the clamped kidney, with or without inhibition of NFAT5, were detected by TUNEL assay and quantitated by laser scanning cytometry. The number of apoptotic cells in the OM increased nearly 2-fold after knockdown of NFAT5 (%TUNEL to PI: EGFP-U6-N5-ex8; 34.5 ± 6.3, EGFP-U6; 19.7 ± 2.4, p<0.05). These data are consistent with H&E staining showing exacerbation of histological features including loss of brush border, flattened tubular epithelium, tubular dilation, tubular lumen debris, and interstitial inflammatory cell infiltration after knockdown of NFAT5. Collectively, the data suggest that NFAT5 is part of a protective mechanism in the TAL and OM that limits renal damage induced by IRI, and may attenuate long-term detrimental renal effects associated with IRI.