Abstract：Objective To study the optimal fermentation conditions for lipstatin production by S. toxytricini FIM-17-16. Methods The single factor experiments and orthogonal experiments were adopted to optimize the liquid fermentation medium and parameters of S. toxytricini FIM-17-16. Results The optimum fermentation medium and parameters were sunflower seed oil 7%, glycerine 2%, soybean flour 3.25%, bran 1%, ZnSO4 0.01%, lecithin 1.00% and CaCO3 0.08%, initial pH not adjusted, filling volume 80mL for each 500mL shake flask, cultivation time of seed 20h, inoculum size 15%, shaker’s rotating speed 230r/min, fermentation temperature 28℃, and fermentation cycle 168h. Compared with that using the original fermentation conditions, the production of lipstatin reached 9,667μg/mL under the optimization of fermentation conditions, which increased 23%, and the fermentation cycle shortened by 24h. Conclusion After optimizing the fermentation conditions, the ability of S. toxytricini FIM-17-16 to biosynthesize lipstatin has been greatly improved, laid the foundation for further industrial fermentation.