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Valproic acid (VPA), a histone deacetylase inhibitor, has been shown to affect cell growth and differentiation in various in vitro and in vivo models. The aim of this study is to evaluate the effects of VPA on viability and osteogenic differentiation of mesenchymal stem cells derived from the human gingival tissue.Stem cells derived from the gingiva were grown in the presence of VPA at concentrations ranging from 0.125 to 8 mM. Cell morphology was assessed on days 3, 5, and 7, and cell proliferation was analyzed on the same days using a Cell Counting Kit-8 (CCK-8). Alizarin Red-S staining was used to assess differentiation of the stem cells.The control group showed a normal fibroblast morphology when cultured in growth media. The shape of cells in the 8 mM group was more flat than cells in other groups, and fewer cells were present. A statistically significant decrease in cell proliferation was seen in the 8 mM group. Results of Alizarin Red-S staining showed a significant decrease in mineralization in the 8 mM group.Taken together, this study demonstrated that VPA, at the tested concentrations, decreases the viability of stem cells derived from the human gingiva. The decreases in osteogenic differentiation were achieved via the decrease of Rux2 expression. The concentration and application time of VPA treatment should be meticulously controlled to minimize any detrimental effects.