We studied the role of classical phagocytic NADPH oxidase (Nox) in the pathogenesis of kidney allograft tubulointerstitial fibrosis. Immunofluorescence studies showed that Nox-2 and p22phox (electron transfer subunits of Nox) colocalized in the tubulointerstitium of human kidney allografts. Tubular Nox-2 also colocalized with α-SMA in areas of injury, suggestive of epithelial-to-mesenchymal transition (EMT). Interstitial macrophages (CD68+) and myofibroblasts (α-SMA+) expressed Nox-2 while graft infiltrating T cells (CD3+) and mature fibroblasts (S100A4+) were Nox-2−. These results were confirmed in the Fisher-to-Lewis rat kidney transplant model. Areas of tubulitis were associated with Nox-2 and α-SMA, suggestive of EMT. Immunoblot analyses showed that Nox-2 upregulation was associated with oxidative stress (nitrotyrosine) and fibrogenesis (α-SMA and phospho-Smad2) at 3 weeks and 6 months. Allografts treated with Nox inhibitors (DPI or apocynin) for 1 week showed reduced fibronectin and phospho-Smad2 and increased E-cadherin levels. Cyclosporine A, TGF-β1 and angiotensin II increased Nox-2 mRNA levels 2- to 7-fold in vitro (NRK52E cells). Treatment with specific Nox inhibitors (DPI or apocynin) prevented the downregulation of E-cadherin and upregulation of fibronectin transcripts. In aggregate, these studies suggest that Nox-2 is involved in the pathogenesis of allograft tubulointerstitial fibrosis via activation transcription factor Smad2, EMT and myofibroblasts.