Polyamines are multifunctional cationic amines that are indispensable for cellular proliferation and of key significance in the growth of rapidly regenerating tissues. Given that the hair follicle (HF) is one of the most highly proliferative organs in mammalian biology, it is not surprising that polyamines are crucial to HF growth. Although the role of polyamines in hair growth and function is well established, the exact role of spermidine in the human HF and its mechanisms of action remain largely unknown. Therefore, we have utilized a serum-free human HF organ cultures to determine the role of spermidine on HFs in situ. Normal human anagen VI scalp HFs were microdissected and organ-cultured with spermidine (0.1-1 µM). Direct application of spermidine led to increased hair shaft elongation, accompanied by prolongation of anagen duration (the growth phase of the HF). In addition, a tendency towards increased proliferation of hair matrix keratinocytes was observed. Quantitative immunohistomorphometry also revealed that spermidine upregulated protein expression of K15 and K19, the epithelial stem cell-associated keratins, and changed K15-promoter activity in situ, thus implicating a novel role for spermidine in regulating stem cell function. These experiments provide first evidence that spermidine is a potent stimulator of human hair growth and keratin expression under human HF organ culture conditions, and argues that a renaissance in polyamine research in hair biology, well beyond the inhibition of polyamine synthesis in hirsutism therapy, is important for the development of novel therapeutic strategies for the manipulation of human hair growth.