The objectives of this study were to determine the effects of deoxyshikonin on lymphangiogenesis. Deoxyshikonin enhanced the ability of human dermal lymphatic microvascular endothelial cells (HMVEC-dLy) to undergo time-dependent in vitro cord formation. Interestingly, an opposite result was observed in cells treated with shikonin. The increased cord formation ability following deoxyshikonin treatment correlated with increased VEGF-C mRNA expression to higher levels than seen for VEGF-A and VEGF-D mRNA expression. We also found that deoxyshikonin regulated cord formation of HMVEC-dLy by increasing the HIF-1 α mRNA level, HIF-1 α protein level, and the accumulation of HIF-1 α in the nucleus. Knockdown of the HIF-1 α gene by transfection with siHIF-1 α decreased VEGF-C mRNA expression and cord formation ability in HMVEC-dLy. Deoxyshikonin treatment could not recover VEGF-C mRNA expression and cord formation ability in HIF-1 α knockdown cells. This indicated that deoxyshikonin induction of VEGF-C mRNA expression and cord formation in HMVEC-dLy on Matrigel occurred mainly via HIF-1 α regulation. We also found that deoxyshikonin promoted wound healing in vitro by the induction of HMVEC-dLy migration into the wound gap. This study describes a new effect of deoxyshikonin, namely, the promotion of cord formation by human endothelial cells via the regulation of HIF-1 α . The findings suggest that deoxyshikonin may be a new drug candidate for wound healing and treatment of lymphatic diseases.