Illigera aromatica was fermented by Clonostachys rogersoniana. The acetylcholinesterase (AChE) inhibitory effects of unfermented and fermented I. aromatica revealed that C rogersoniana-fermented I. aromatica (CFIA) induced significantly more AChE inhibitory activity (IC50: 35.4 +/- 2.1 mu g/mL). The biotransformation of actinodaphnine (1) into (4R,6aS)4-hydroxyactinodaphnine (2) was found during the fermentation, which played an important role in the improvement of the AChE inhibitory activity of L aromatica. Subsequently, the fermentation conditions including the solid-liquid ratio, fermentation temperature, and fermentation time-were optimized. I. aromatica immersed in 100-200% water and fermented with C. rogersoniana at ambient temperature for 30 days was conducive to the biotransformation of actinodaphnine (1) and improved the AChE inhibitory activity of I. aromatica. The present study provides a novel approach for improving the pharmacological effect of L aromatica and suggests that CFIA may be used as an alternative AChE inhibitor. (C) 2019, The Society for Biotechnology, Japan. All rights reserved.