Increasing evidence suggests that various neurotrophins and neuropeptides play an important role in the progression of hair follicle cycling. Among them, nerve growth factor (NGF) and substance P (SP) have attracted special interest recently. However, the interaction between these factors during hair cycling has not yet been systematically studied. We therefore investigated the mutual relationships between NGF and SP and the mechanism by which the anagen stage of the hair cycle is initiated. Fluctuations in numbers of SP-positive nerve fibers and variations in amounts of SP, NGF, and another neurotrophic factor, glial cell-derived neurotrophic factor, in skin in the C57BL/6 mouse depilation-induced hair cycle model, together with the spatiotemporal expression patterns of each of these factors, were followed simultaneously by enzyme-linked immunosorbent assay and immunohistochemistry. The main finding was that a surge in NGF expression and a rapid increase in NGF content in skin is an initial event within 1 day after depilation, followed by elevation of SP content and numbers of SP-containing fibers 2 days after the increase in NGF. Our findings suggest that a rapid and abundant increase in NGF plays a key role in the induction and progression of anagen hair cycling through keratinocyte growth promotion. NGF may also induce plastic changes such as sprouting and hyperplasia in dermal nerve fibers and enhance their SP production. Elevated levels of SP in skin may additionally contribute to the progression of consecutive anagen hair cycles.