A significant difference in the bond distances or angles and short contacts between the two adjacent isoalloxazine (IA) rings in the riboflavin tetracarboxylate crystal are attributable to the nonbonded interaction. The perturbation of the electronic state in the IA ring accompanied by the elimination of the protic solvent well explains an occurrence of charge transfer (CT) interaction. This inference is supported by the fact that the IA rings are overlapping face to tail in the concentrated solution and the solvation takes place at the IA ring. In the frontier molecular orbital theory, it is possible to consider that the interaction occurs between the HOMO of the solvent-eliminated IA, which acts as a donor, and the LUMO of the solvated IA, which behaves as an accepter. The effect of the CT interaction on the stacking interaction can be measured quantitatively as the red shift or quenching in the reflection and the emission spectra and the signal in the ESR. It is certain that this report gives an important clue about the interrelation between the CT interaction and the hydrophobic environment in the stacking interaction.