The role of ion rotation in determining ion mobilities is explored using the subtle gas phase ion mobility shifts based on differences in ion mass distributions between isotopomer ions that have been observed with ion mobility spectrometry (IMS) measurements. These mobility shifts become apparent for IMS resolving powers on the order of ∼1500 where relative mobilities (or alternatively momentum transfer collision cross sections; Ω) can be measured with a precision of ∼10 ppm. The isotopomer ions have identical structures and masses, differing only in their internal mass distributions, and their Ω differences cannot be predicted by widely used computational approaches, which ignore the dependence of Ω on the ion's rotational properties. Here, we investigate the rotational dependence of Ω, which includes changes to its collision frequency due to thermal rotation as well as the coupling of translational to rotational energy transfer. We show that differences in rotational energy transfer during ion-molecule collisions provide the major contribution to isotopomer ion separations, with only a minor contribution due to an increase in collision frequency due to ion rotation. Modeling including these factors allowed for differences in Ω to be calculated that precisely mirror the experimental separations. These findings also highlight the promise of pairing high-resolution IMS measurements with theory and computation for improved elucidation of subtle structural differences between ions.