Matrix metalloproteinases (MMPs) are endopeptidases that degrade extracellular matrix and involved in ischemic organ injuries. The present study was designed to determine the role of MMP-2 in the development of ischemic acute kidney injury (AKI). AKI was induced in MMP-2 wild-type (MMP-2(+/+)) mice by 30, 60, 90, and 120 min renal ischemia and reperfusion. Renal histology, expression and activity of MMP-2 and MMP-9, and renal function were examined during the development of AKI. AKI was also induced in MMP-2-deficient (MMP-2(-/-)) mice and MMP-2(+/+) mice treated with inhibitor of MMPs (minocycline and synthetic peptide MMP inhibitor). In MMP-2(+/+) mice, MMP-2 and MMP-9 activities increased significantly at 2 to 24 h, peaked at 6 h, after reperfusion. Immunohistochemical analysis identified MMP-2 in the interstitium around tubules and peritubular capillaries in the outer medulla. Acute tubular injury (ATI), including apoptosis and necrosis, was evident in the outer medulla at 24 h, along with renal dysfunction. As ischemia period increases, MMP-2 and MMP-9 activities at 6 h and severity of AKI at 24 h increased depending on the duration of ischemia between 30 and 120 min. However, the kidneys of MMP-2(-/-) mice showed minimal ATI; serum creatinine 24 h after reperfusion was significantly low in these mice. Inhibitors of MMPs reduced ATI and improved renal dysfunction at 24 h. We conclude that MMPs, especially MMP-2 have a pathogenic role in ischemia-reperfusion AKI, and that inhibitors of MMPs can protect against ischemic AKI.