Brefeldin A, a macrolide lactone antibiotic, can block the secretory process in eukaryotic cells by interfering in the endoplasmic reticulum to Golgi membrane traffic, resulting in the disassembly of Golgi apparatus and redistribution of Golgi-apparatus proteins into the endoplasmic reticulum, contributing to its antifungal, antiviral, anticancer activities.Although significant advances have been made in brefeldin A synthesis, commercial brefeldin A production exclusively relies on microbial fermentation.So far, Phyllosticta medicaginis was reported to afford a maximal brefeldin A titer of approximately 300 mg 1-1.In this work, a brefeldin A-overproducing fungus, Eupenicillium brefeldianum ZJB082702 (CCTCC M 208113), was successfully bred from an endophytic fungus E.brefeldianum A1163.A promising brefeldin A batch fermentation process was established with E.brefeldianum ZJB082702, yielding brefeldin A at 1304.7 mg 1-1.Since few documents concerning brefeldin A separation and purification are available, macroporous resin adsorption chromatography was evaluated for the application in brefeldin A recovery from the fermentation broth of E.brefeldianum ZJB082702.Through experimental optimization of column adsorption and desorption, brefeldin A in purity of 90.4% (w/w), 92.1% (w/w) yield was recovered by a one-step macroporous resin adsorption chromatography, using a stepwise elution protocol.As crystallization is a powerful separation process, complementary to chromatography, and can mass-produce antibiotics with excellent purities, cooling crystallization, evaporative crystallization and isothermal antisolvent crystallization were examined for brefeldin A purification in terms of crystallization yield, crystal shape, size, and distribution.Among them, isothermal antisolvent crystallization was preferred.Outcomes revealed that the optimum isothermal antisolvent crystallization conditions were as follows: operation temperature 303K, agitation speed 80 r/min, seeding mass 2.0% (w/v), antisolvent pure water fed into the saturated brefeldin A ethanol solution within 2.0h at a constant flow rate to a final volumetric fraction of 2.0, crystal growth time of 2.0h.The resultant first batch crystals were further subjected to recrystallization in acetone using evaporative crystallization, yielding colorless prism crystals with purity over 99.0% (w/w).Therefore, an efficient brefeldin A manufacturing process was successfully developed, enabling brefeldin A production at a competitive cost.