After oral mucosal injury, the healing response following specific steps that lead to wound closure and to tissue repair. Multiple cell populations are involved in this process; in particular, fibroblasts play a key role in the production of extracellular matrix (ECM). During wound healing the remodeling of ECM is a key stage to restore the tissue functionality through multifunctional fibroblast populations that are placed in the connective tissues of gingiva and periodontal ligament. Notably, a fibroblast sub-type (myofibroblast) is centrally involved in collagen synthesis and fibrillar remodeling. The present work evidenced the role of Transforming Growth Factor-beta1 (TGF-β1) to mediate human gingival fibroblasts (hGFs) differentiation into myofibroblasts derived from gingival fibroblasts (myo-hGFs). The morphological and functional features were analyzed through Confocal Laser Scanning Microscopy (CLSM), flow cytometry, and western blotting analyses. The specific markers, such as alpha-Smooth Muscle Actin (α-SMA), Vimentin, E-cadherin, β-catenin, and Smad 2/3, were modulated in myo-hGFs after the induction with TGF-β1, at different time points (24, 48, and 72 h). After 72 h of treatment TGF-β1 operates as an inducer of hGFs into myo-hGFs differentiation. We propose that TGF-β1 may promote in vitro the fibroblasts-to-myofibroblasts transition via the morphological and molecular modifications, as the induction of α-SMA, Vimentin, E-cadherin, β-catenin, and Smad 2/3.Keywords: differentiation; fibroblast; gingival; myofibroblast; transforming growth factor-beta1.