Our previous works showed chronic infection of Mycoplasma hyorhinis (M. hyorhinis) was associated with gastric cancer metastasis, but the mechanisms were unknown. Herein, we found M. hyorhinis induced epithelial-mesenchymal transition (EMT) in gastric cancer cell MGC803, which was counteracted by inhibitor of NF-κB signaling or p65 knockdown. Furthermore, we found that TLR4 associated with p37, a membrane protein of M. hyorhinis. Knock-down or inhibition of TLR4 antagonized M. hyorhinis-induced NF-κB signaling, EMT, and cell migration. Thus, M. hyorhinis induces EMT and promotes cell migration via TLR4-NF-κB signaling, which provides a clue to the pathogenesis of M. hyorhinis in gastric cancer.