Pancreatic cancer (PC) is one of the most lethal solid malignancies and new therapeutic strategies are urgently needed. β1,3-N-acetylglucosaminyltransferase V (B3GNT5) may be a potential option for cancer treatment, but its role in pancreatic cancer remains unknown. We systematically verified the regulatory role of B3GNT5 on the malignant progression of pancreatic cancer and found that B3GNT5 overexpression significantly enhanced the malignant behaviors of tumor cells, such as invasion, migration, proliferation, stem cell properties, and drug resistance. In order to clarify the possible targets and molecular regulatory mechanisms of B3GNT5, we collected control and B3GNT5 overexpressing pancreatic cancer cells, analyzed the differential proteins by Label-free quantitative proteomics analysis. The Gene Ontology enrichment analysis and the Kyoto Encyclopedia of Genes and Genomes were used to interpret the proteomic data. Finally, AKT1, MAPK1, and MAPK3 were identified as possible downstream targets of B3GNT5, which provides a more comprehensive perspective for understanding B3GNT5 aggravation of pancreatic cancer progression.