To investigate the extracted process of gossypol acetic acid (G-AA) from cottonseed soapstock and explore the improvement of its yield and purity, a novel ultrasound-assisted extraction and crystallization method was introduced to this process. Under the optimized conditions, preliminary G-AA with the yield of 1300 mg and the purity of 95.9% could be obtained from 100 g of fresh soapstock by ultrasound-assisted extraction. In addition, UV, IR, and NMR spectrum further confirmed the detailed chemical structure of G-AA. Assay of inhibiting human prostate tumor cell line PC-3 and human breast cancer cell line MDA-MB-231 revealed its biological activity, the values of IC50 are 9.096 mu mol/L and 14.37 mu mol/L respectively. In comparison with the conventional solvent extraction, this novel process increases the content of G-AA over 90%, reduces the little of crystallization by 75%, and retains the anticancer activity of gossypol. (c) 2009 American Institute of Chemical Engineers AIChE J, 55: 797-806, 2009