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Introduction: Keratinocyte stem cells, residing in the basal layer of the epidermis, are thought to function in the process of the epidermis renewal and maintain the barrier property of the skin. Objective: This study has explored for the first time that vanillin, a natural compound from vanilla cured beans, has the ability to augment the stem cell property and signaling in the human HaCaT keratinocytes. Methods: The cell viability effect of vanillin on the keratinocytes was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The Western blot analysis was performed to determine the level of stem cell-mediated proteins, mesenchymal, and epithelial markers. Results: Results showed that treatment of the HaCaT cells with vanillin at non-toxic concentrations was able to significantly up-regulate the stemness mediators Oct-4, p-Oct-4 and Nanog. Besides, we investigated the mesenchymal and epithelial makers and found that vanillin could increase the expression of epithelial adhesive protein E-cadherin but has no significant effect on mesenchymal mediator slug. Conclusions: As the stem cells, as well as their stemness properties, hold the central functions of skin renewal and repair, information gain from this study may benefit the development of vanillin to be used for skin therapy. [ABSTRACT FROM AUTHOR]