Human periodontal ligament stem cells (hPDLSCs) are potential seed cells for bone tissue engineering, but the molecular regulatory mechanisms of their multi-differentiation remain unclear. Here, we found that Yes-associated protein (YAP), a transcriptional coactivator in Hippo signaling pathway, regulated the multi-differentiation ability of hPDLSCs: overexpressing YAP contributed to an enhancement of osteogenic differentiation and a decrease in adipogenic differentiation, while knocking down YAP inhibited the osteogenic differentiation and promoted the adipogenic differentiation of hPDLSCs. In addition, YAP promoted the stabilization and nuclear transfer of β-catenin in hPDLSCs, probably through regulating several upstream proteins of the Wnt/β-catenin signaling pathway, including LRP6 and DVL3. Treatment with DKK1 or Wnt3a reversed the effects of overexpressing or knocking down YAP on non-phospho β-catenin (stabilized β-catenin) and cell differentiation. Taken together, YAP promoted osteogenic and inhibited adipogenic differentiation of hPDLSCs in vitro, which was partly via LRP6 and DVL3 mediated Wnt/β-catenin signaling pathway. YAP could be a candidate regulatory target for facilitating the application of hPDLSCs in bone regeneration.