Cell migration crucial contributor to metastasis, a critical process associated with the mortality of cancer patients. The initiation of metastasis is triggered by epithelial-mesenchymal transition (EMT), along with the changes of the expression of EMT marker proteins. Inflammation plays a significant role in carcinogenesis and metastasis. Lipopolysaccharide (LPS), a typical inflammatory agent, promoted the generation of superoxide through the activation of p-Tyr42 RhoA, Rho-dependent kinase 2 (ROCK2) and the phosphorylation of p47phox. In addition, p-Tyr42 RhoA activated phospholipase D1 (PLD1), with PLD1 and phosphatidic acid (PA) being involved in superoxide production. PA also regulated the expression of EMT maker proteins. Consequently, we have identified MHY9 (Miosin IIA, NMIIA) as a PA-binding protein in response to LPS. MYH9 also contributed cell migration and the alteration of the expression of EMT marker proteins. P-Tyr42 RhoA, PLD1 and MYH9 associated to form a complex, which was distributed in both the cytosol and nucleus. In addition, we have found that p-Tyr42 RhoA and PLD1 associated with the ZEB1 promoter. Taken together, we propose that p-Tyr42 RhoA and PLD1, responsible for producing PA, and PA-bound MYH9 are involved in the regulation of ZEB1 expression, thereby promoting cell migration.