Early invasive growth and metastasis are features of pancreatic cancer that rely on its resistance to anoikis, an apoptosis program activated on loss of matrix anchorage. How anoikis is regulated is unclear. UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine-kinase (GNE) was silenced, or p16 was overexpressed, in human pancreatic carcinoma cells. Gene expression profiling, enzymatic assays, Western blotting, and cell cycle analysis were conducted. Silencing of GNE, the key enzyme of sialic acid biosynthesis, sensitizes pancreatic cancer cells to anoikis. Accordingly, we observed a loss of GNE enzyme activity in cells, which became anoikis susceptible after transfection with the tumor suppressor p16. Similarly, studies of another cell line with low GNE activity revealed strong anoikis susceptibility, confirming the association of low GNE activity and anoikis susceptibility. Gene expression profiling demonstrated that the loss of GNE triggered the transcriptional activation of the ATF4-ATF3-CHOP pathway, leading to apoptosis in the framework of the unfolded protein response. In silico analysis showed that GNE up-regulation occurred predominantly in pancreatic cancer but also in other malignancies. Delineation of GNE-dependent signaling pathways may provide targets that control anchorage dependence and/or restore drug efficacy, which is of utmost relevance for the treatment of pancreatic cancer.