Extracellular matrix overexpression is a common final pathway that leads to ventricular remodeling. Fibronectin plays a pivotal role in this progress. In the work presented here, we explored the possibility of direct inhibition of fibronectin synthesis in rat cardiac fibroblasts by small interference RNA (siRNA). We found that siRNA could successfully suppress the fibronectin overexpression stimulated by angiotensin II. To overcome the limitations of plasmid-based siRNA, we subcloned the H1 promoter into pLXIN, a commercially available retroviral vector. We found that H1 promoter worked very well to form the small hairpin RNA (shRNA) on the retroviral vector, and the fibronectin expression was dramatically down regulated by shRNA. We think the retroviral shRNA delivery system that we have constructed may have potential roles in treating ventricular remodeling.