We report the site-specific incorporation of a thiocyanate vibrational probe into the active site oxyanion hole of ketosteroid isomerase (KSI) to test the effect of hydrophobic steroid binding and solvent exclusion on the local electrostatic environment at this position. While binding of an uncharged ground state steroid analog shifts the observed –CN vibrational frequency by +0.4 cm−1 relative to unliganded KSI, binding of an intermediate steroid analog containing localized negative charge results in a +2.8 cm−1 shift. Based on a Stark tuning rate of 0.7 cm−1/(MV/cm), this shift indicates a fivefold larger change in the projection of the local electric field along the –CN bond in the presence of the charged ligand. Binding of a single ring phenolate with oxyanion charge localization equivalent to the intermediate steroid analog but lacking distal hydrocarbon rings results in an identical –CN peak shift. We conclude that solvent exclusion and replacement by hydrophobic steroid rings negligibly alter the electrostatic environment within the KSI oxyanion hole. Development of localized negative charge analogous to that of the dienolate intermediate during steroid isomerization dramatically increases the magnitude of the local electric field. This increase reflects field contributions from the localized negative charge itself as well as possible increased ordering of active site dipoles in response to charge localization.