Background: Rapidly regenerating tissues need sufficient polyamine synthesis. Since the hair follicle (HF) is a highly proliferative mini-organ, polyamines may also be important for normal hair growth. However, the role of polyamines in human HF biology and their effect on HF epithelial stem cells in situ remains largely unknown. Methods and Findings: We have studied the effects of the prototypic polyamine, spermidine (0.1-1 μM), on human scalp HFs and human HF epithelial stem cells in serum-free organ culture. Under these conditions, spermidine promoted hair shaft elongation and prolonged hair growth (anagen). Spermidine also upregulated expression of the epithelial stem cell-associated keratins K15 and K19, and dose-dependently modulated K15 promoter activity in situ and the colony forming efficiency, proliferation and K15 expression of isolated human K15-GFP+ cells in vitro. Inhibiting the rate-limiting enzyme of polyamine synthesis, ornithine decarboyxlase (ODC), downregulated intrafollicular K15 expression. In primary human epidermal keratinocytes, spermidine slightly promoted entry into the S/G2-M phases of the cell cycle. By microarray analysis of human HF mRNA extracts, spermidine upregulated several key target genes implicated e.g. in the control of cell adherence and migration (POP3), or endoplasmic reticulum and mitochondrial functions (SYVN1, NACA and SLC25A3). Excess spermidine may restrict further intrafollicular polyamine synthesis by inhibiting ODC gene and protein expression in the HF's companion layer in situ. Conclusions: These physiologically and clinically relevant data provide the first direct evidence that spermidine is a potent stimulator of human hair growth and a previously unknown modulator of human epithelial stem cell biology.