Tilmicosin is a semi-synthetic macrolide derivative of tylosin. The production process of tilmicosin involved extraction using butyl acetate and subsequent stripping using an aqueous buffer solution. In this work, the effect of pH on the solubility of tilmicosin in its aqueous phase was examined. It was found that the solubility of tilmicosin is much higher at a low pH than at a high pH due to its Lewis base nature. The apparent distribution coefficients of tilmicosin between the aqueous and butyl acetate phases were measured at different pH and temperature values. The results demonstrated that the purification of tilmicosin could be carried out by solvent extraction at a high pH value and stripping using an aqueous buffer solution at a low pH value. Based on the dissociation mechanism of tilmicosin in water, the intrinsic distribution coefficient K-0 between the aqueous and solvent phases and two dissociation equilibrium constants pK(1) and pK(2) were correlated as K-0 = 81.55, p K-1 = 8.67, p K-2 = 6.55, respectively. The dissociation activation energy was found to be -46.5 kJ/mol at pH 4.5. This high activation energy value was mostly attributed to the formation of tilmicosin salt.