Reactive oxygen species (ROS) derived from NOX4/NADPH oxidase are known to play important roles in tissue fibrogenesis. However, signaling pathways downstream of NOX4 involved in fibrogenesis have not yet been well established. A proteomics technique with cleavable isotope-coded affinity tags was applied to TGF-β-stimulated IMR-90 cells derived from human lung fibroblasts. In cells transfected with siRNAs against NOX4, we found that levels of endoglin, a co-receptor of TGF-β, were reproducibly decreased compared with those in control siRNA-transfected cells. Quantitative PCR and Western blotting revealed that the expression level of endoglin was decreased at protein levels by knock-down of NOX4. Bafilomycin A1, an inhibitor of lysosome, disturbed down-regulation of endoglin by anti-NOX4-siRNAs. In addition, an inhibitor of protein kinase C (PKC), GF109203X, as well as a pseudosubstrate inhibitor of atypical PKC, suppressed the reduction in endoglin induced by anti-NOX4 siRNAs. These findings suggested the involvement of atypical PKC in degradation of endoglin upon depletion of NOX4. Thus, NOX4-derived ROS may stabilize endoglin by regulating atypical PKC.