Wound healing is often delayed in the patients whose sensory and autonomic innervation is impaired. We hypothesized that existence of neurites in the skin may promote wound healing by inducing differentiation of fibroblasts into myofibroblasts with consequent wound contraction. In the current study, we examined the effect of neurons on differentiation of fibroblasts and contraction of collagen matrix in vitro using a new co-culture model. Neuronal cell line, PC12 cells, of which the neurite outgrowth can be controlled by adding nerve growth factor, was used. Rat dermal fibroblasts were co-cultured with PC12 cells extending neurites or with PC12 cells lacking neurites. Then, differentiation of fibroblasts into myofibroblasts and contraction of the collagen matrix was evaluated. Finally, we examined whether direct or indirect contact with neurites of PC12 cells promoted the differentiation of fibroblasts. Our results showed that fibroblasts co-cultured with PC12 extending neurites differentiated into myofibroblasts more effectively and contracted the collagen matrix stronger than those with PC12 lacking neurites. Direct contact of fibroblasts with neurites promoted more differentiation than indirect contact. In conclusion, direct contact of fibroblasts with neuronal processes is important for differentiation into myofibroblasts and induction of collagen gel contraction, leading to promotion of wound healing.