7 alpha,15 alpha-diOH-DHEA is a key precursor of the novel oral contraceptive Yasmin. Colletotrichum lini could catalyze dehydroepiandrosterone (DHEA) at the 7 alpha and 15 alpha positions. In this work, C. lini resting cells were applied in the bioconversion of DHEA to 7 alpha,15 alpha-diOH-DHEA. In the presence of 2 % (w/v) Tween-80, the conversion efficiency of DHEA increased drastically. The DHEA conversion and the 7 alpha,15 alpha-diOH-DHEA yield increased by 34.6 and 87.0 %, respectively, at the DHEA concentration of 10 g/L. Furthermore, the effects of Tween-80 on substrate solubility and C. lini physiological properties were studied. Results showed that the DHEA solubility with 2 % Tween-80 increased by 7.8 times. Meanwhile, the mycelia were integrated and full in the presence of 2 % Tween-80. The analysis on fatty acid profile of the C. lini cell membrane indicated that Tween-80 increases the content of unsaturated fatty acid. All above results suggested that the enhanced product yield caused by Tween-80 was mainly associated with easier substrate-molecule transportation across the cell membrane of C. lini.