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Background: Dermal fibroblasts play a pivotal role in hair follicle regeneration during wound repair. Recently, dermal fibroblast‐conditioned medium (DFCM), which contains multi‐peptide factors (MPFs), has been used to promote wound repair. Aim: This study aimed to investigate the stimulatory effects of MPF‐containing DFCM on hair growth. Methods: MPF‐containing DFCM was prepared using human neonatal dermal fibroblasts. Outer root sheath (ORS) and dermal papilla (DP) cells were cultured in MPF‐containing DFCM. We examined the expression and secretion of growth factors and cytokines using quantitative polymerase chain reaction and a growth factor array. In addition, the effect of MPFs on β‐catenin activity was determined using the TOPflash assay. All experiments were repeated at least three times with separate batches of cells. Results: MPF‐containing DFCM increased keratinocyte growth factor (KGF), vascular endothelial growth factor (VEGF), and epidermal growth factor (EGF) mRNA expression in ORS cells and KGF and VEGF mRNA expression in DP cells. When ORS cells were treated with MPF‐containing DFCM, the secretion of several growth factors, including EGF, VEGF, insulin‐like growth factor‐binding protein (IGFBP)‐4, IGFBP‐6, and fibroblast growth factor‐7, was increased in the cell‐cultured medium compared with that in control. Additionally, MPF‐containing DFCM increased the transcriptional activation of β‐catenin in DP cells. Conclusions: These results suggest that MPF‐containing DFCM might stimulate hair growth by inducing growth factors in ORS and DP cells and regulating β‐catenin in DP cells. [ABSTRACT FROM AUTHOR]