Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most hypoxic and subsequently glycolytic tumors. Thus the inhibition of glycolysis appears to be an attractive therapeutic approach that should be explored. Methods: A series of 2-deoxy analogs of D-glucose, namely, 2-deoxy-D-glucose (2-DG), 2-fluoro-D-glucose (2-FG), 2-chloro-D-glucose (2-CG), 2-bromo-D-glucose (2-BG), and 2,2-difluoro-D-glucose (2,2-diFG) were synthesized in our laboratory. The ability of 2-deoxy-2-halo-D-glucose derivatives to inhibit glycolysis in pancreatic cancer cell lines was tested using Seahorse 96 analyzer and XF Glycolysis Stress Test kit. The cellular ATP level was measured using CellTiter-Glo assay. Viability of cells cultured in normoxic and hypoxic conditions were assessed using MTS assay. Results: 2,2-diFG and 2-FG were significantly more potent inhibitors than 2-DG of the Extracellular Acidification Rate (ECAR) of medium in oligomycin-stimulated PDAC cells (complete inhibition at <12.5 mM, whereas, the 100 mM is a commonly used concentration for 2-DG in Seahorse 96 experiments). Interestingly, 2-bromo (2-BG) and 2-chloro (2-CG) analogs have a limited or no effect on glycolysis at tested concentration. Consistently, both, 2,2-diFG and 2-FG, inhibited proliferation of pancreatic cancer cell lines more potently than 2-DG in either normoxic and hypoxic conditions, with the differential effects being significantly more pronounced under hypoxia. Conclusion: Both, 2,2-diFG and 2-FG have a superior to 2-DG ability to inhibit glycolysis in vitro and should be further evaluated in vivo in the PDAC models as a potential anticancer agents. Citation Format: Rafal Zielinski, Izabela Fokt, Aleksandra Rusin, Stanislaw Skora, Waldemar Priebe. 2-Deoxy-2-halo-D-glucose derivatives inhibition of glycolysis in pancreatic cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 50.