Cancer cell metabolic reprogramming includes a shift in energy production from oxidative phosphorylation to less efficient glycolysis even in the presence of oxygen (Warburg effect) and use of glutamine for increased biosynthetic needs. This necessitates greatly increased glucose and glutamine uptake, both of which enter the hexosamine biosynthetic pathway (HBP). The HBP end product UDP-N-acetylglucosamine (UDP-GlcNAc) is used in enzymatic post-translational modification of many cytosolic and nuclear proteins by O-linked β-N-acetylglucosamine (O-GlcNAc). Here, we observed increased HBP flux and hyper-O-GlcNAcylation in human pancreatic ductal adenocarcinoma (PDAC). PDAC hyper-O-GlcNAcylation was associated with elevation of OGT and reduction of the enzyme that removes O-GlcNAc (OGA). Reducing hyper-O-GlcNAcylation had no effect on non-transformed pancreatic epithelial cell growth, but inhibited PDAC cell proliferation, anchorage-independent growth, orthotopic tumor growth, and triggered apoptosis. PDAC is supported by oncogenic NF-κB transcriptional activity. The NF-κB p65 subunit and upstream kinases IKKα/IKKβ were O-GlcNAcylated in PDAC. Reducing hyper-O-GlcNAcylation decreased PDAC cell p65 activating phosphorylation (S536), nuclear translocation, NF-κB transcriptional activity, and target gene expression. Conversely, mimicking PDAC hyper-O-GlcNAcylation through pharmacological inhibition of OGA suppressed suspension culture-induced apoptosis and increased IKKα and p65 O-GlcNAcylation, accompanied by activation of NF-κB signaling. Finally, reducing p65 O-GlcNAcylation specifically by mutating two p65 O-GlcNAc sites (T322A and T352A) attenuated the induction of PDAC cell anchorage-independent growth. Our data indicate that hyper-O-GlcNAcylation is anti-apoptotic and contributes to NF-κB oncogenic activation in PDAC. Background: Cancer cells rely on energy metabolism that requires increased glucose uptake and constitutive NF-κB activity for survival. Results: Pancreatic cancer cells display elevated O-GlcNAcylation, reduction of which inhibits cell survival and oncogenic NF-κB signaling. Conclusion: Hyper-O-GlcNAcylation is anti-apoptotic and contributes to NF-κB activation in pancreatic cancer. Significance: Targeting hyper-O-GlcNAcylation may serve as a novel therapeutic intervention in pancreatic cancer.