Objective：To explore the expression of β-galactoside-α-2，3-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：Bioinformatic analysis， qRT-PCR，and immunohistochemistry（IHC）were employed to measure ST3GAL3 mRNA and protein expression levels in ESCC and adjacent normal tissues，followed by correlation analysis with patient prognosis and clinicopathological data. The expression of ST3GAL3 gene in different ESCC cell lines was analyzed through the CCLE database. We established ST3GAL3-WT and its catalytic site mutant overexpression，as well as ST3GAL3 knockdown and its rescue cells. We assessed cell proliferation，migration，and spreading abilities on ECM using cell proliferation assay and Transwell assay，respectively. The effect of ST3GAL3 on the expression of proteins related to adhesion signaling and the α-2，3-sialylation of Integrin α5 and β1catalyzed by ST3GAL3 was detected by Western blot and lectin-immunoprecipitation（Lectin-IP），respectively. Results：The mRNA expression levels of ST3GAL3 were significantly higher in the ESCC tissues than in the ajacent normal tissues（P ＜ 0.01），with concurrent elevated protein levels in tumor samples. High expression of ST3GAL3 in ESCC was positively correlated with patient poor prognosis and was associated with TNM staging （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 α - 2，3 - sialylation of Integrin α5 and β1. Conclusion：The expression of ST3GAL3 was increased in ESCC tissues，and its high expression was positively correlated with patients’poor prognosis，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.