Objective: To explore the expression of β-galactoside-α2,3-sialyltransferase-3 (ST3GAL3) in esophageal squamous cell carcinoma (ESCC) and its relationship with patient prognosis and correlation with clinicopathologic characteristics, as well as to investigate the function and molecular mechanism of ST3GAL3 in cell proliferation and migration. Methods: The mRNA and protein expression of ST3GAL3 in ESCC and paired normal tissues were examined by GEO database, qRT-PCR and immunohistochemistry (IHC), respectively. Additionally, the relationship between ST3GAL3 expression and patient prognosis, as well as its correlation with clinicopathological characteristics, was analyzed. We analyzed the expression of ST3GAL3 gene in different ESCC cell lines by CCLE database, and established ST3GAL3-WT and its catalytic site mutant overexpression, as well as ST3GAL3 knockdown and its rescue cells. The cell proliferation, migration and spreading abilities on ECM was assessed using cell proliferation assay and Transwell assay, respectively. The effect of ST3GAL3 on the expression of proteins related to adhesion signaling and the α2,3-sialylation of Integrin α5 and β1catalyzed by ST3GAL3 was detected by Western blot (WB) and lectin-immunoprecipitation (Lectin-IP), respectively. Results: The mRNA expression level of ST3GAL3 was significantly higher in the ESCC tissues than in the normal tissues (P < 0.01). The protein expression level of ST3GAL3 was significantly upregulated in the ESCC specimens compared with the paired paracancer tissues, and the high expression of ST3GAL3 in ESCC was positively correlated with patient poor prognosis and TNM stage (P = 0.004), T classification (P < 0.001), and lymph node metastasis (P = 0.017). ST3GAL3 promoted ESCC cell migration on ECM in a catalytic function-dependent manner. Furthermore, ST3GAL3 mediated cell spreading and adhesion signaling under ECM-coating conditions. Moreover, ST3GAL3 catalyzed the α2,3-sialylation of Integrin α5 and β1. Conclusion: The expression of ST3GAL3 was increased in the ESCC, and its high expression was positively correlated with patient poor prognosis and TNM stage, T classification, and lymph node metastasis. ST3GAL3 enhanced ESCC cell spreading and adhesion signaling on ECM, therefore promoting ECM-mediated cell migration, possibly through the α-2,3 sialylation of adhesion receptors such as Integrin α5 and β1.