Conditioned medium from adipose-derived stem cells (ADSCs) stimulates both collagen synthesis and migration of dermal fibroblasts. However, it is still unknown whether conditioned media from tumor growth factor (TGF)-β1-treated ADSCs (TGF-β1-treated ADSCs-CM) induces increased expression of type I collagen, matrix metalloproteinase-1 (MMP-1), and migration as well as cell cycle regulatory proteins in fibroblasts, compared to non-treated ADSCs-CM. Our data showed that TGF-β1-treated ADSCs-CM promoted effectively the proliferation and migration of human skin fibroblasts, compared to non-treated ADSCs-CM. In addition the expression of MMP-1 were markedly increased by treatment of TGF-β1-treated ADSCs-CM in fibroblasts, compared to non-treated ADSCs-CM. Expression of type I collagen protein were slightly increased by treatment of TGF-β1-treated ADSCs-CM in fibroblasts. The expression of cell cycle regulators of G1/S phase transition were not markedly altered by treatment of TGF-β1-treated ADSCs-CM. Finally, artificial wounds were made using a 4-mm punch biopsy in hairless mice and TGF-β1-treated ADSCs-CM were injected into the wound area. The injection of TGF-β1-treated ADSCs-CM promoted the wound healing process in hairless mice. Taken together, our data indicated that TGF-β1-treated ADSCs-CM induced up-regulation of type I collagen and MMP-1, promoted the migration of skin fibroblasts, and thereby promoted the wound healing process in vivo. Our data indicate that TGF-β1-treated ADSCs-CM will be a component for a wound healing accelerating agent.