Epithelial cell function is modulated by mechanical forces imparted by the extracellular environment. The transmission of forces onto the cytoskeleton by modalities such as mechanical stress and matrix stiffness is necessary to address by the development of new experimental models that permit finely tuned cell mechanical challenges. Herein, we developed an epithelial tissue culture model, named the 3D Oral Epi-mucosa platform, to investigate the role mechanical cues in the epithelial barrier. In this platform, low-level mechanical stress (0.1 kPa) is applied to oral keratinocytes, which lie on 3D fibrous collagen (Col) gels whose stiffness is modulated by different concentrations or the addition of other factors such as fibronectin (FN). Our results show that cells lying on intermediate Col (3 mg/mL; stiffness = 30 Pa) demonstrated lower epithelial leakiness compared with soft Col (1.5 mg/mL; stiffness = 10 Pa) and stiff Col (6 mg/mL; stiffness = 120 Pa) gels, indicating that stiffness modulates barrier function. In addition, the presence of FN reversed the barrier integrity by inhibiting the interepithelial interaction via E-cadherin and Zonula occludens-1. Overall, the 3D Oral Epi-mucosa platform, as a new in vitro system, will be utilized to identify new mechanisms and develop future targets involved in mucosal diseases. © 2023 by the authors.