Enamel matrix derivative, obtained from developing porcine teeth, is composed mainly of amelogenin proteins and used topically in periodontal surgery for advanced periodontitis to regenerate lost connective tissues. The primary objective of this study was to investigate the effects of enamel matrix derivative on skin wound healing. Secondly, in vitro effects of enamel matrix derivative on dermal fibroblasts and microvascular endothelial cells were examined. Full-thickness, circular 2-cm skin wounds in white 16-week-old rabbits were treated thrice weekly with enamel matrix derivative (30 mg/ml) in the vehicle propylene glycol alginate or with vehicle alone. Enamel matrix derivative treatment increased the amount of granulation tissue and accelerated time to complete epithelialization by 3 days (p < 0.001) compared to vehicle treatment. In cultured fibroblasts, vascular endothelial growth factor levels in conditioned media were increased more than fivefold (p < 0.001) with enamel matrix derivative treatment (0.1mg/ml) over control, measured by specific enzyme-linked immunosorbent assay. Enamel matrix derivative also increased release of matrix metalloproteinase-2 more than threefold from fibroblasts (p < 0.001) and from endothelial cells (p < 0.001). Thus, enamel matrix derivative significantly accelerated wound closure in rabbits, possibly by increasing levels of growth factors and proteinases important for granulation tissue formation and remodeling.