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• The nucleation behavior of tiamulin hydrogen fumarate methanol solvate (THFMS) was investigated. • The solubility and cosolvency effect of THFMS were studied in methanol-ethyl acetate binary mixtures. • The interfacial energy, the critical nucleation potential, and the critical nucleation parameterswere discussed. To investigate the nucleation behavior of tiamulin hydrogen fumarate methanol solvate (THFMS), the solubility, induction time, and metastable zone width (MSZW) of THFMS in methanol-ethyl acetate binary mixtures were measured. The nucleation potential model, which is based on the classical nucleation theory and correlates the induction time and MSZW, has been employed to deal with the experimental data and estimate the interfacial energy and the critical nucleation potential. The estimated values determined from the nucleation potential model are in accordance with the experimental values. The interfacial energy and the critical nucleation potential of THFMS steadily decrease with the increasing saturation temperature, whereas those of THFMS initially decrease and subsequently increase as the ethyl acetate content increases. The results demonstrate that an increase in saturation temperature leads to enhanced nucleation favorability, while the difficulty of nucleation initially decreases and then increases with increasing ethyl acetate content. Furthermore, the critical nucleation parameters, including the critical Gibbs free energy, the critical nucleus size, and the critical number of molecules in a nucleus, exhibit the same trend in alterations regarding both saturation temperature and ethyl acetate content. [ABSTRACT FROM AUTHOR]