In this article, macro-porous resin, SP825, was used as adsorbent for the adsorption separation of erythromycin A (EA) and erythromycin C (EC). The effect of solution polarity and temperature on adsorption of EA and EC with their mixture was determined by batch adsorption experiments in detail. Adsorption isotherms were modeled by competitive Langmuir equations, selectivity coefficients were obtained, and thermodynamic parameters such as changes of Gibbs free energy, enthalpy, and entropy were calculated by van't Hoff relationships. Then, dynamic adsorption and desorption separation experiments with the appropriate solution polarity and temperature obtained from the batch experiments were examined in a fixed-bed. Breakthrough curves were determined to understand the different dynamic adsorption performances for EA and EC. After the adsorption and desorption separation, the mass concentration ratio of EA to EC increased sharply from 9:1 to 25.3:1.