Cardiac fibrosis is a leading cause of heart failure, particularly heart failure with preserved ejection fraction (HFpEF). Currently, there is no effective treatment for HFpEF. 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 was demonstrated when cardiac fibroblasts were exposed to homogenates from wild-type H9c2. On the other hand, increased proliferation was significantly attenuated in cardiac fibroblasts exposed to homogenates from Nox1-disrupted H9c2. In Nox1-disrupted H9c2 cells, the expression of osteoglycin (Ogn), and podocan (Podn), which are small leucine-rich proteoglycans and known to regulate cardiac remodeling, were up-regulated. When homogenates from Nox1-disrupted H9c2 with disruption of Ogn or Podn exposed to cardiac fibroblasts, the proliferation of fibroblasts was significantly restored compared to those exposed to Nox1-disrupted H9c2. These findings suggest that NOX1 promotes cardiac fibrosis via down-regulation of Ogn or Podn in cardiomyocytes.