Bitespiramycin was purified by selective adsorption. The adsorption and desorption properties of bitespiramycin on six different macroporous resins (HZ806, HZ816, HZ820, HZ830, XAD16, and SP207) were compared systematically. According to the adsorption capacity and selectivity towards 4 ''-O-isovalerylspiramycin, HZ820 was chosen as the most suitable resin for bitespiramycin purification. The equilibrium data on HZ820 in a batch system were well described by Langmuir and Freundlich models. The film and pore diffusion model was successfully measured in batch adsorption kinetics. Dynamic adsorption and desorption experiments were also performed using a packed column of HZ820 to optimize the separation process of 4 ''-O-isovalerylspiramycin from aqueous solution. After being treated with HZ820, the 4 ''-O-isovalerylspiramycin content increased from 80.4% to 91.7%, and the 4 ''-O-isovalerylspiramycin III content increased from 41.7% to 64.4%.