Background: EPS8L1, an analog of epidermal growth factor receptor pathway substrate 8 (Eps8), was screened out in our previous work from clinical samples of patients with ovarian cancer. Our studies also indicated that EPS8L1 might involve in various biological activities. In this study, we further investigated the effect and mechanism of EPS8L1 on the migration and metastasis of ovarian cancer. Methods: SKOV-3 cells with EPS8L1 overexpression and knockdown were established to perform in vitro scratch healing, transwell assay and actin-staining studies. Ovarian cancer mice with lung metastasis was established. Bioinformatics assay, qRT-PCR and Western blot were conducted to identify correlated proteins. Also, the Rac1 activity and the expression of MAPK pathway-related proteins were evaluated. Result: The knockdown of EPS8L1 inhibited the cellular migration in vitro and reduced tumors colonization in vivo. Actin-staining and ELISA experiment suggested that EPS8L1 regulated actin formation and cytoskeleton remodeling. Besides, mRNA and protein expression confirmed that EPS8L1 regulated the downstream molecule T-cell lymphoma invasion and metastasis 2 (TIAM2) and stimulated the activation of Rac1. Also, the phosphorylation levels of P38, Erk and Jnk in MAPK pathway decreased after EPS8L1 knockdown. Conclusion: The upregulation of EPS8L1 could promote migration and metastasis of ovarian cancer cells by regulating cytoskeleton remodeling. The mechanism underlying might be EPS8L1 regulates TIAM2 to induce Rac-GDP to Rac-GTP and then activates the downstream MAPK pathway. As a regulatory gene in cell migration and metastasis, EPS8L1 probably provide a new therapeutic target for ovarian cancer treatment.