Objective This study aimed to investigate the differentiation of human periodontal fibroblasts into myofibroblasts induced by transforming growth factor(TGF)-β1 in vitro and the time-dependent effect of induced myofibroblast(MFB). Methods Human periodontal fibroblasts were cultured with 5 μg•L-1 TGF-β1 for 72 h in vitro to induce to MFB. As an MFB marker, alpha-smooth muscle actin(α-SMA) was examined through immunocytochemistry and immunofluorescence. After induction, MFB was harvested at 12, 24, 48, 72, 96, and 120 h. Cell activity was evaluated through flow cytometry and immunocytochemistry. Results Treatment with 5 μg•L-1 TGF-β1 induced positive α-SMA expression. MFB maintained its phenotype after 120 h, and cell activity was stabilized after 72 h. Conclusion MFB was successfully induced in vitro with 5 μg•L-1 TGF-β1. The cell phenotype was also stabilized within 72 h.