In order to discover the steroid biotransformation ability of ?lamentous fungus Aspergillus niger TCCC41650, we studied the fermentation of 4-androstene-3,17-dione with A. niger TCCC41650. The transformation product was purified, crystallized and determined as 16β-hydroxy-androst-4-ene-3,17-dione by X-ray single crystal diffraction method. The best fermentation condition was found to be pH 6.0, ethanol amount 2% with a substrate concentration of 1‰, the transformation rate is 85.81% after 72 h. Based on the best of our knowledge, 16β-hydroxylation rarely occurs in microbial transformations of steroid. This study laid the foundation for the research of 16β-hydroxylation steroids.