Decorin is a natural transforming growth factor-β1 (TGF-β1) antagonist. Reduced decorin synthesis is associated with dermal scarring, and increased decorin expression appears to reduce scar tissue formation. To investigate the therapeutic potential of decorin for keloids, human dermal fibroblasts (HDFs) and keloid-derived fibroblasts (KFs) were transduced with decorin-expressing adenovirus (dE1-RGD/GFP/DCN), and we examined the therapeutic potential of decorin-expressing Ad for treating pathologic skin fibrosis. Decorin expression was examined by immunofluorescence assay on keloid tissues. HDFs and KFs were transduced with dE1-RGD/GFP/DCN or control virus, and protein levels of decorin, epidermal growth factor receptor (EGFR) and secreted TGF-β1 were assessed by Western blotting and ELISA. And type I and III collagen, and matrix metalloproteinase-1 (MMP-1) and matrix metalloproteinase-3 (MMP-3) mRNA levels were measured by real-time RT-PCR. Additionally, we immunohistochemically investigated the expression levels of the major extracellular matrix (ECM) proteins in keloid spheroids transduced with dE1-RGD/GFP/DCN. Lower decorin expression was observed in the keloid region compared to adjacent normal tissues. After treatment with dE1-RGD/GFP/DCN, secreted TGF-β1 and EGFR protein expressions were decreased in TGF-β1-treated HDFs and KFs. Also, type I and III collagen mRNA levels were decreased, and the expression of MMP-1 and MMP-3 mRNA was strongly upregulated. In addition, the expression of type I and III collagen, fibronectin and elastin was significantly reduced in dE1-RGD/GFP/DCN-transduced keloid spheroids. These results support the utility of decorin-expressing adenovirus to reduce collagen synthesis in KFs and keloid spheroid, which may be highly beneficial in treating keloids.