Exploratory pilot experiments in our lab(s) had raised the possibility that vasoactive intestinal peptide (VIP), a key immunoinhibitory neuropeptide released by perifollicular sensory nerve fibers, may be a novel modulator of human HF pigmentation. This hypothesis was followed-up in the current study. After showing that the VIP receptors, VPAC1 and VPAC2, are expressed at gene and protein level in human anagen hair bulbs, we investigated whether VIP (10-7M) modulates human HF pigmentation in situ and in isolated human HF melanocytes (HFiMc). VIP significantly increased both the number of intraepithelial c-Kit+ HF melanocytes (HFMc) in situ and intrafollicular c-Kit transcription. By contrast, VIP down-regulated SCF gene and protein expression in organ-cultured human HFs. Interestingly, c-Kit/gp100 double- immunostaining indicated that most of the VIP-induced c-Kit+ HFMc represent immature/amelanotic HFMc. Intriguingly, VIP up-regulated the total number of gp100+, MITF+, or p-MITF+ HFMc and promoted intrafollicular melanin production in situ. VIP also significantly stimulated melanogenesis in isolated human HFiMc, suggesting that it impacts directly on HFMc, but also slightly inhibited HFMc/HFiMc proliferation in situ and in vitro. Preliminary results obtained with specific antagonists suggested that VPAC1 and VPAC2 mediated the VIP effects on HFMc. Since microarray and qRT-PCR analyses showed an up-regulation of phosphodiesterase 4D7 transcription in VIP-treated HFs, VIP may regulate the cAMP level in HFMc, possibly by utilizing downstream pathways shared with α-MSH signaling. In summary, our results reveal VIP as a novel, complex and clinically-relevant neuroendocrine regulator of human HF pigmentation.