The biotransformation of cyclopamine (1) and its congeners (2, 3 and 4) by Cunninghamella echinulata (ACCC 30369) was investigated. The chemical structures of two new congeners (2 and 4) and nine new metabolites were elucidated by 1D NMR (H-1, C-13 and DEPT), 2D NMR (COSY, HMBC, HSQC and NOESY) and HRESIMS analyses and further confirmed by X-ray diffraction studies. Among these compounds, 2 and 4 showed moderate cytotoxicity in HepG2 cells (2, IC50 = 8.03 +/- 1.92 mu M) and A549 cells (4, IC50 = 10.19 +/- 2.18 mu M). Conversely, the cytotoxicity of the nine metabolites was sharply reduced. Similar to 1, compound 4 induced a cyclopia phenotype in zebrafish embryos at 20 mM. Moreover, compound 4 was more stable than 1 in an acidic environment. (C) 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.