Effective therapy to prevent organ fibrosis, which is associated with more than half of all mortalities, remains elusive. Involvement of tumor suppressor ataxia telangiectasia mutated (ATM) in the TGF-β1 pathway related to renal fibrosis is largely unknown. ATM activation (pATM(Ser1981)) increased 4-fold in the tubulointerstitial region of the unilateral ureteral obstruction-injured kidney in mice correlating with SMAD3 and p53(Ser15) phosphorylation and elevated levels of p22(phox) subunit of the NADPH oxidases (NOXs), and fibrotic markers, plasminogen activator inhibitor-1 (PAI-1), and fibronectin, when compared to contralateral (contra) or sham controls. In fact, ATM is rapidly phosphorylated at Ser(1981) by TGF-β1 stimulation. Stable silencing and pharmacologic inhibition of ATM ablated TGF-β1-induced p53 activation (>95%) and subsequent PAI-1, fibronectin, connective tissue growth factor, and p21 expression in human kidney 2 (HK-2) tubular epithelial cells and normal rat kidney-49 fibroblasts (NRK-49F). ATM or p53 depletion in HK-2 cells, moreover, bypassed TGF-β1-mediated cytostasis evident in control short hairpin RNA-expressing HK-2 cells. Interestingly, stable silencing of NOX subunits, p22(phox) and p47(phox), in HK-2 cells blocked TGF-β1-induced pATM(Ser1981) (>90%) and target gene induction via p53-dependent mechanisms. Furthermore, NRK-49F fibroblast proliferation triggered by conditioned media from TGF-β1-stimulated, control vector-transfected HK-2 cells decreased (∼ 50%) when exposed to conditioned media from ATM-deficient, TGF-β1-treated HK-2 cells. Thus, TGF-β1 promotes NOX-dependent ATM activation leading to p53-mediated fibrotic gene reprogramming and growth arrest in HK-2 cells. Furthermore, TGF-β1/ATM-initiated paracrine factor secretion by dysfunctional renal epithelium promotes interstitial fibroblast growth, suggesting a role of tubular ATM in mediating epithelial-mesenchymal cross-talk highlighting the translational benefit of targeting the NOX/ATM/p53 axis in renal fibrosis.