Solvent organization is a key but underexploited contributor to the thermodynamics of protein–ligand recognition, with implications for ligand discovery, drug resistance, and protein engineering. Here, we explore the contribution of solvent to ligand binding in the Haemophilus influenzae virulence protein SiaP. By introducing a single mutation without direct ligand contacts, we observed a >1000-fold change in sialic acid binding affinity. Crystallographic and calorimetric data of wild-type and mutant SiaP showed that this change results from an enthalpically unfavorable perturbation of the solvent network. This disruption is reflected by changes in the normalized atomic displacement parameters of crystallographic water molecules. In SiaP’s enclosed cavity, relative differences in water-network dynamics serve as a simple predictor of changes in the free energy of binding upon changing protein, ligand, or both. This suggests that solvent structure is an evolutionary constraint on protein sequence that contributes to ligand affinity and selectivity.