The invention discloses a mesenchymal stem cell for inhibiting tumor growth and a preparation method and an application, and relates to the technical field of the medicine. The provided mesenchymal stem cell for inhibiting the tumor growth is a TLR5 positive mesenchymal stem cell. The preparation method for the TLR5 positive mesenchymal stem cell is to separate TLR5 high-expression positive mesenchymal stem cells by using a flow-type or overexpress TLR5 on the mesenchymal stem cell. The invention further provides a cultural supernatant containing a serum-free medium and the TLR5 positive mesenchymal stem cell, and further provides applications of the TLR5 positive mesenchymal stem cell and the cultural supernatant for preparing an anti-tumor drug. The cultural supernatant in TLR5+MSC conditions has the remarkable inhibiting effects to the in-vitro proliferation, the migration and the clonality of a tumor cell. The TLR5-activated MSC is capable of remarkably improving the in-vitro killing activity of a NK cell. The TLR5-activated MSC is capable of inhibiting the tumor growth in Lewis lung cancer mice.