Cardiac fibrosis is a leading cause of diastolic dysfunction in the failing heart. Currently, there is no effective treatment for diastolic dysfunction. We previously reported that doxorubicin-induced cardiac fibrosis was suppressed in mice deficient in Nox1, a non-phagocytic isoform of superoxide-producing NADPH oxidase. In this study, the role of NOX1 in the development of cardiac fibrosis was investigated in cultured cells using a rat cardiomyoblast cell line H9c2 and cardiac fibroblasts isolated from adult male mice. Increased proliferation of cardiac fibroblasts induced by transforming growth factor-β (10 ng/ml) or fetal bovine serum (1%) was significantly suppressed when cells were exposed to homogenates from Nox1-diurupted H9c2, but not from wild-type cells. In Nox1-disrupted H9c2 cells, expression of Collagen4a1 (Col4a), Osteoglycin (Ogn), and Podocan (Podn) were up-regulated. When cardiac fibroblasts were exposed to homogenates from Nox1- as well as Col4a -, Ogn -, or Podn -disrupted H9c2 cells, proliferation of fibroblasts was significantly restored compared to those exposed to Nox1-disrupted cells. These findings suggest that NOX1 promotes cardiac fibrosis via down-regulation of fibrosis inhibitory factors in cardiomyocytes.