We recently described the binding of 1-β-phosphonomethylene-1-deoxy-d-glucopyranose, (S)-1-β-phosphonofluoromethylene-1-deoxy-D-glucopyranose (βG1CF_SP), and (R)-1-β-phosphonofluoromethylene-1-deoxy-d-glucopyranose (βG1CF_RP) to the enzyme β-phosphoglucomutase as transition state analogues of phosphoryl transfer through formation of stable MgF₃⁻ and AlF₄⁻ complexes (Proc. Natl. Acad. Sci. U.S.A. 2014, 111, 12384). Herein, we describe in detail the five-fold difference in acidity (pK_a2) for the (S)- and (R)-configured diastereomeric fluorophosphonates through a series of NMR spectroscopy experiments. The differences in acidity were corroborated using computational quantum mechanical calculations to determine structures of lowest energy conformers and provide insight into why the (S) isomer is substantially more acidic.