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Background Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy with distinct tumor biology that drives rapid progression and poor therapeutic response. A prominent feature of PDAC is abnormal glycosylation, especially hypersialylation, which involves excessive sialic acid expression and contributes to tumor growth and immune evasion. Sialyltransferases (STs) mediate hypersialylation; however, the specific enzymes involved in PDAC progression remain unclear. This study aims to identify and characterize key sialyltransferases in PDAC.Methods We integrated RNA-seq data and clinical information from PDAC patients obtained from TCGA and GTEx databases. A six-gene prognostic model based on STs expression was developed using LASSO regression and validated across independent PDAC cohorts. Bioinformatic analyses evaluated associations between ST expression and survival outcomes, immune infiltration, immune checkpoint expression, tumor mutational burden, and drug sensitivity. Single-cell RNA sequencing and multiplex immunofluorescence (mIHC) were employed to investigate interactions between ST-expressing tumor cells and immune cells. Functional validation was performed thourgh in vitro knockdown experiments to assess the effects of key ST(s) on CA19-9 synthesis in PDAC cells.Results The STs-based prognostic model showed strong predictive power (AUC=0.797), with ST3GAL1 contributing most significantly. ST3GAL1 was markedly overexpressed in PDAC compared to normal tissue and was associated with worse overall survival (HR=1.45, p<0.001). High ST3GAL1 expression was linked to an immunosuppressive tumor microenvironment characterized by reduced CD8+ T cells, elevated neutrophil infiltration, and increased M2 macrophages. Single-cell analysis and mIHC confirmed a positive correlation between ST3GAL1+ tumor cells and neutrophil infiltration. Notably, ST3GAL1-driven α2,3-sialylation was critical for CA19-9 biosynthesis. Knockdown of ST3GAL1 in PDAC cell lines significantly decreased CA19-9 expression, highlighting its critical role in generating this key tumor biomarker Additionly, the knockdown of ST3GAL1 observably suppressed proliferation and migration in CFPAC-1 and Panc 05.04 cells.Conclusions ST3GAL1 is a key driver of hypersialylation in PDAC and serves as an independent prognostic biomarker and potential therapeutic target. Its overexpression reshapes the immunosuppressive microenvironment. Moreover, ST3GAL1 regulates t CA19-9 biosynthesis, a critical glycan biomarker for PDAC diagnosis and monitoring., These findings highlight ST3GAL’s value in improving prognostic stratification and identifying patients resistant to immunotherapy. Targeting ST3GAL1-mediated sialylation may offer a novel strategy to overcome current challenges in PDAC.