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Outer root sheath (ORS) is a highly proliferative component of a hair follicle. This study is performed to investigate whether hypoxia-induced elevation of hypoxia-inducible factor (HIF)-1α, a transcriptional activator, contributes to the outgrowth of ORS cells in vitro.Hair follicles with intact ORS collected from 4-month old male American minks were cultured in normoxic or hypoxic condition (3% oxygen) for 7 days. Primary ORS cells isolated from the mink hair follicles were exposed to hypoxia for 12, 24 or 48 h, and their proliferation was analyzed with immunofluorescence assay using anti-proliferating cell nuclear antigen (PCNA) antibody. The migratory ability of ORS cells was detected via the transwell chamber. The endogenous HIF-1α was knocked down with its specific siRNA in ORS cells.Hypoxic exposure induced an elevation of HIF-1α in ex vivo cultured hair follicles. The mRNA and protein levels of sonic hedgehog (Shh), Shh receptor Patched 1, Smoothened and glioma-associated oncogene homologue 1 were upregulated. In vitro, hypoxia induced an increase in HIF-1α in ORS cells. Further, under hypoxic condition, the number of PCNA-positive cells was increased, and more cells migrated towards high serum media. Hypoxia-enhanced proliferation and migration of ORS cells were suppressed either by HIF-1α siRNA or by pharmacological inhibitors of Shh pathway, cyclopamine and GANT61. The activation of Shh pathway was attenuated in HIF-1α-silenced ORS cells under hypoxic condition.Our work demonstrates a direct role of activated HIF-1/Shh biological axis in sustaining the development of ORS in vitro.