This study reports the sugaring-out extraction of erythromycin from fermentation broth using acetonitrile (ACN) as solvent and glucose as a mass separating agent. Different process parameters--glucose concentration, temperature, ACN/water ratio and pH--were optimized to achieve maximum extraction of erythromycin. 88% (w/w) of erythromycin was extracted from the model system with following optimized conditions: glucose 156.3 g/L; temperature 4 °C; ACN/water ratio 1 and pH 8.3. Further, the effect of typical fermentation media components (starch, soybean flour, CaCO3, NaCl and (NH4)2SO4) on sugaring out extraction of erythromycin was also investigated. Starch, soybean flour and CaCO3 were observed to affect erythromycin extraction only at higher concentration. Removal of suspended solids from simulated as well as real broth prior to extraction enhanced the extraction efficiency (from 72% to 87%). Sugaring out extraction of erythromycin was found to be more effective than salting out extraction. Also, higher partition coefficient was achieved in the present work than other reported methods using carbohydrates as mass separating agent. Further, it was found that the antimicrobial activity of erythromycin was preserved during sugaring out extraction of erythromycin.