Hepatocellular carcinoma (HCC) patients are at high risk for both local recurrence and distant metastasis and tightly associated with poor prognosis. Exploring the molecular mechanism will provide a new opportunity in developing personal treatment for advanced HCC patients. As a critical member of the Retinal Determination Gene Network (RDGN), EYA1 has been identified as a tumor promoter in various cancers; however, its role in HCC has never been investigated. The present study was aimed to explore the role of EYA1 in HCC development. By analyzing public microarray datasets, we found that the EYA1 mRNA level was enhanced in HCC, which was significantly correlated with an aggressive phenotype and poor prognosis. Besides, EYA1 was coordinated with the fibronectin type III domain containing 36 (FNDC3B) to promote the migration and invasion of HCC cells. Western blot assays indicated that EYA1 not only increased the abundance of FNDC3B but also contributed to Epithelial-Mesenchymal Transition (EMT)-like phenotype change, like increased N-cadherin and decreased E-cadherin expression. Collectively, this study suggested that EYA1 activated FNDC3B to promote the migration and invasion in HCC. The aberrant expressions of EYA1 and FNDC3B may become the poor predictors for HCC patients.