Tissue inhibitors of metalloproteinases (TIMPs) are endogenous inhibitors of matrix metalloproteinases (MMPs). While TIMP2 and TIMP3 inhibit MMPs, TIMP3 also inhibits activation of pro-MMP2, whereas TIMP2 promotes it. Here we assessed the differential role of TIMP2 and TIMP3 in renal injury using the unilateral ureteral obstruction model. Gene microarray assay showed that post obstruction, the lack of TIMP3 had a greater impact on gene expression of intermediate, late injury- and repair-induced transcripts, kidney selective transcripts, and solute carriers. Renal injury in TIMP3(-/-), but not in TIMP2(-/-), mice increased the expression of collagen type I/III, connective tissue growth factor, transforming growth factor-β, and the downstream Smad2/3 pathway. Interestingly, ureteral obstruction markedly increased MMP2 activation in the kidneys of TIMP3(-/-) mice, which was completely blocked in the kidneys of TIMP2(-/-) mice. These changes are consistent with enhanced renal tubulointerstitial fibrosis in TIMP3(-/-) and its reduction in TIMP2(-/-) mice. The activities of tumor necrosis factor-α-converting enzyme, caspase-3, and mitogen-activated kinases were elevated in the kidneys of TIMP3(-/-) mice but not TIMP2(-/-) mice, suggesting enhanced activation of apoptotic and pathological signaling pathways only in the obstructed kidney of TIMP3(-/-) mice. Thus, TIMP2 and TIMP3 play differential and contrasting roles in renal injury: TIMP3 protects from damage, whereas TIMP2 promotes injury through MMP2 activation.