Separations using CE employing non-aqueous BGE are already as well established as separations in aqueous buffers. The separation mechanisms in achiral CE with nonaqueous BGEs are most likely similar to those in aqueous buffers. However, for the separation of enantiomers involving their interaction with chiral buffer additives, the interaction mechanisms might be very different in aqueous and non-aqueous BGEs. While the hypothesis regarding distinct mechanisms of enantiomer separations in aqueous and non-aqueous BGEs has been mentioned in several papers, no direct proof of this hypothesis has been reported to date. In the present study, the enantiomers of propranolol were resolved using CE in aqueous and non-aqueous methanolic BGEs with two single isomer sulfated derivatives of beta-CD, namely heptakis (2,3-diacetyl-6-sulfo)-beta-CD and heptakis (2,3-dimethyl-6-sulfo)-beta-CD. The enantiomer migration order of propranolol was inverted when an aqueous BGE was replaced with non-aqueous BGE in the case of heptakis (2,3-dimethyl-beta-sulfo)-beta-CD but remained the same in the case of heptakis (2,3-diacetyl-6-sulfo)-beta-CD. The possible molecular mechanisms leading to this reversal of enantiomer migration order were studied by using nuclear overhauser effect spectroscopy in both aqueous and non-aqueous BGEs.