Objective　and　design:　Pancreatic　cancer　is　a　highly　malignant　tumor　that　is　well　known　for　its　poor　prognosis.　Based　on　glycosylation,　we　performed　integrated　quantitative　N-glycoproteomics　to　investigate　the　synergistic　anti-tumor　effects　of　aspirin　and　gemcitabine　on　pancreatic　cancer　cells　and　explore　the　potential　molecular　mechanisms　of　chemotherapy　in　pancreatic　cancer.　Methods　and　results:　Two　pancreatic　cancer　cell　lines　(PANC-1　and　BxPC-3)　were　treated　with　gemcitabine,　aspirin,　and　a　combination　(gemcitabine　+　aspirin).　We　found　that　the　addition　of　aspirin　enhanced　the　inhibitory　effect　of　gemcitabine　on　the　activity　of　PANC-1　and　BxPC-3　cells.　Quantitative　N-glycoproteome,　proteome,　phosphorylation,　and　transcriptome　data　were　obtained　from　integrated　multi-omics　analysis　to　evaluate　the　anti-tumor　effects　of　aspirin　and　gemcitabine　on　pancreatic　cancer　cells.　Mfuzz　analysis　of　intact　N-glycopeptide　profiles　revealed　two　consistent　trends　associated　with　the　addition　of　aspirin,　which　showed　a　strong　relationship　between　N-glycosylation　and　the　synergistic　effect　of　aspirin.　Further　analysis　demonstrated　that　the　dynamic　regulation　of　sialylation　and　high-mannose　glycoforms　on　ECM-related　proteins　(LAMP1,　LAMP2,　ITGA3,　etc.)　was　a　significant　factor　for　the　ability　of　aspirin　to　promote　the　anti-tumor　activity　of　gemcitabine　and　the　drug　resistance　of　pancreatic　cancer　cells.　Conclusions:　In-depth　analysis　of　N-glycosylation-related　processes　and　pathways　in　pancreatic　cancer　cells　can　provide　new　insight　for　future　studies　regarding　pancreatic　cancer　therapeutic　targets　and　drug　resistance　mechanisms.　©　2023,　Springer　Nature　Switzerland　AG.