Biological modification of natural products is an important approach to improve the pharmacological properties. 4'-d-beta-Demethylepipodophyllotoxin (4'-d-beta-DMEP), a typical natural lignin, is used as the aglycon of topoisomerase inhibitor etoposide (VP-16). In this study, 4'-d-beta-DMEP was isomerized into the novel product 4'-d-alpha- DMEP with 43.09% conversion by P. purpurogenum ACCC 32170. 4'-d-alpha-DMEP was largely produced extracellularly in the late fermentation stage, which was accompanied with the decrease of dry cell weight and the increase of culture pH. A constitutively expressed cytosolic enzyme was involved in the transformation and functioned when culture pH ranged from 7.0-10.0. The IC50 value of 4'-d-alpha-DMEP was around 0.80 mu M against tumor cells MCF-7, which was significantly reduced by 11.27 and 17.34 times than 4'-d-beta-DMEP (i.e., 9.82 mu M) and VP-16 (i.e., 14.67 mu M). This study provided the novel podophyllotoxin derivative with higher anti-tumor activity and microbial transformation process, which would promote transferring lignans into the clinic trial.