Glycogen phosphorylase is a molecular target for the design of potential hypoglycemic agents. Structure-based design pinpointed that the 3′-position of glucopyranose equipped with a suitable group has the potential to form interactions with enzyme’s cofactor, pyridoxal 5′-phosphate (PLP), thus enhancing the inhibitory potency. Hence, we have investigated the binding of two ligands, 1-(β-d-glucopyranosyl)5-fluorouracil (GlcFU) and its 3′-CH2OH glucopyranose derivative. Both ligands were found to be low micromolar inhibitors with Ki values of 7.9 and 27.1 μm, respectively. X-ray crystallography revealed that the 3′-CH2OH glucopyranose substituent is indeed involved in additional molecular interactions with the PLP γ-phosphate compared with GlcFU. However, it is 3.4 times less potent. To elucidate this discovery, docking followed by postdocking Quantum Mechanics/Molecular Mechanics – Poisson–Boltzmann Surface Area (QM/MM-PBSA) binding affinity calculations were performed. While the docking predictions failed to reflect the kinetic results, the QM/MM-PBSA revealed that the desolvation energy cost for binding of the 3′-CH2OH-substituted glucopyranose derivative out-weigh the enthalpy gains from the extra contacts formed. The benefits of performing postdocking calculations employing a more accurate solvation model and the QM/MM-PBSA methodology in lead optimization are therefore highlighted, specifically when the role of a highly polar/charged binding interface is significant.