In this work, we discuss the challenging time-resolved fluorescence anisotropy of subtilisin Carlsberg (SC), which contains a single Trp residue and is a model fluorescence system. Experimental decay rates and quenching data suggest that the fluorophore should be exposed to water, but the Trp is partially buried in a hydrophobic pocket in the crystallographic structure. In order to study this inconsistency, molecular dynamics simulations were performed to predict the anisotropy decay rates and emission wavelengths of the Trp. We confirmed the inconsistency of the crystallographic structure with the experimentally observed fluorescence data and performed free energy calculations to show that the buried Trp conformation is 2 orders of magnitude (∼3 kcal/mol) more stable than the solvent-exposed one. However, molecular dynamics simulations in which the Trp side chain was restricted to solvent-exposed conformations displayed a maximum Trp emission wavelength shifted toward lower energies and decay rates compatible with the experimentally probed rates. Therefore, if the solvent-exposed conformations are the most important emitters, the experimental anisotropy can be compatibilized with the crystallographic structure. The most likely explanation is that the fluorescence of the most probable conformation in solution, observed in the crystal, is quenched, and this is consistent with the low quantum yield of Trp113 of SC. Additionally, some experiments might have probed denatured or lysed SC structures. SC anisotropy provides an interesting target for the study of fluorescence anisotropy using simulations, which can be used to test and exemplify how modeling can aid the interpretation of experimental data in a system where structure and solution experiments appear to be inconsistent.