Mesenchymal stem cells (MSCs) have self-renewal ability and the potential for multi-directional differentiation, and their clinical application has promising prospects, but improving the migration ability of MSCs in vivo is one of the challenges. We previously determined mechanical stretch at 1 Hz with 10 % strain for 8 h can significantly promote MSC migration, however, the molecular mechanism remains poorly understood. Here, we reported that the expression and activity of yes-associated protein (YAP) are upregulated after mechanical stretch. As a classical inhibitor of the YAP-TEAD activity and YAP protein, the treatment of verteporfin (VP) suppressed mechanical stretch-promoted MSC migration. We also observed F-actin polymerization after mechanical stretch. Next, we used Latrunculin A (Lat A), the most widely used reagent to depolymerize actin filaments, to treat MSCs and we found that Lat A treatment inhibits MSC migration by suppressing YAP expression and activity. In addition, the protein expression of Piezo1 was also upregulated after mechanical stretch. Knockdown of Piezo1 suppressed mechanical stretch-promoted MSC migration by restraining F-actin polymerization. Together, these findings demonstrate the role of Piezo1/F-actin/YAP signaling pathway in MSC migration under mechanical stretch, providing new experimental evidence for an in-depth understanding the mechanobiological mechanism of MSC migration.