Keloids are defined as overgrowths of scar tissue resulting from abnormal wound healing. They are characterized by excessive dermal deposition of thick, hyalinized collagen bundles resulting from an imbalance between the production and degradation of extracellular matrix (ECM) components. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are two important regulators of ECM degradation and remodeling. To evaluate the role played by knockdown of TIMPs in keloid formation, we transduced human keloid-derived fibroblasts (KFs) with small interfering RNAs targeting TIMP-1 or -2 (siTIMP-1 or siTIMP-2) using a lentiviral vector and assessed the biological effects. We found that MMP-1/TIMP-1 and MMP-1/TIMP-2 complexes were suppressed and that MMP-2 activity was upregulated in KFs expressing siTIMP-1 or siTIMP-2. In addition, increased degradation of collagen type I was observed in the supernatant of KFs expressing siTIMP-1, but not siTIMP-2, with the suppression of cell viability and induction of apoptosis. These results suggest that targeting TIMP-1 using small interfering RNA has significant therapeutic potential as an approach to treating keloids through degradation of their thick collagen bundles.