C-2 Fluorinated castanospermines have been synthesized from a well-protected aldehyde precursor and evaluated as glycosidase inhibitors in comparison with castanospermine, 1-epi-castanospermine and C-1 fluorinated castanospermines. While C-1 fluorinated castanospermines lose nearly all the glycosidase inhibition shown by castanospermine and 1-epi-castanospermine, C-2 fluorinated derivatives of castanospermine were found to be potent and highly specific α-glucosidase inhibitors; however, the C-2 fluorinated 1-epi-castanospermines showed a sharp decrease in inhibition towards all tested enzymes. Docking calculations attributed the sharp decrease of glycosidase inhibition of C-1 fluorinated castanospermines to the disappearance of hydrogen bonds between the original C-1 hydroxyls and residues Arg-526 and Asp-327. The retained potent and specific α-glucosidase inhibition of C-2 fluorinated castanospermines was achieved by the fluorine-induced reestablishment of the docking mode in the active site; and the sharply decreased inhibition of C-2 fluorinated 1-epi-castanospermines can be attributed to obvious binding distorsion and disappearance of the hydrogen bonding with residues His-600 and Arg-526. Reliability of the docking results was evaluated by Molecular Dynamics (MD) simulation, which provided necessary calibrations to the calculation results. The interaction modes of fluorine reported herein are different from the "mimic effect" of fluorine for hydrogen, offering insights and extending our previous work on fluorinated casuarines. These results would be important for the development of castanospermine-related drug candidates for the treatment of diabetes, viral infections and Pompe disease.