Purpose: The underlying mechanism of how topographic cues of artificial scaffolds regulate cell function remains poorly understood. Yes-associated protein (YAP) and β-catenin signaling have both been reported to play important roles in mechano-transduction and dental pulp stem cells (DPSCs) differentiation. We investigated the effects of YAP and β-catenin in spontaneous odontogenic differentiation of DPSCs induced by topographic cues of a poly(lactic-co-glycolic acid) (PLGA) membrane. Methods: The topographic cues and function of a fabricated PLGA scaffold were explored via scanning electron microscopy (SEM), alizarin red staining (ARS), reverse transcription-polymerase chain reaction (RT-PCR), and pulp capping. Immunohistochemistry (IF), RT-PCR, and western blotting (WB) were used to observe the activation of YAP and β-catenin when DPSCs were cultured on the scaffolds. Further, YAP was inhibited or overexpressed on either side of the PLGA membrane, and YAP, β-catenin, and odontogenic marker expression were analyzed using IF, ARS, and WB. Results: The closed side of the PLGA scaffold promoted spontaneous odontogenic differentiation and nuclear translocation of YAP and β-catenin in vitro and in vivo compared to the open side. The YAP antagonist verteporfin inhibited β-catenin expression, nuclear translocation, and odontogenic differentiation on the closed side, but the effects were rescued by LiCl. YAP overexpressing DPSCs on the open side activated β-catenin signaling and promoted odontogenic differentiation. Conclusion: The topographic cue of our PLGA scaffold promotes odontogenic differentiation of DPSCs and pulp tissue through the YAP/β-catenin signaling axis.