Tautomers are structural isomers that readily interconvert and may exhibit different properties. The effect of solvent on tautomeric equilibria in solution has been a subject of some research. Tautomer solvate is less common, and the role of solvent in the crystallization of tautomer solvate remains an interesting topic. In this work, we used 6-amino-1,3-dimethyl-5-nitrosouracil (NAU) as the tautomeric model material, which can present in nitrone–enamine form (Tautomer A) or oxime–imine form (Tautomer B). A solvate with NAU/DMSO ratio of 1:1 was discovered and characterized using single/powder X-ray diffraction and thermogravimetry. The crystal structure of NAU·DMSO was determined for the first time, where only Tautomer A was formed in the tautomeric crystal. Quantum chemical calculation and molecular dynamics simulation were conducted to determine the tautomeric form in DMSO solution. Electrostatic potential analysis, radial distribution function analysis, and binding energy suggested possible DMSO–NAU interaction modes and stable tautomer complexes in solution. Tautomer A-containing complexes were found to dominate in solution, as verified by comparing predicted and experimental 1 H NMR spectra. Findings reveal that the hydrogen bonding between DMSO and NAU is similar in solution and in NAU–DMSO solvate crystal, which helps preserve the form of Tautomer A during solvate crystallization.