Cephalosporin C (CPC) fermentation by Acremonium chrysogenum is an extremely high oxygen-consuming process and oxygen transfer rate in a bioreactor directly affects fermentation performance. In this study, fluid dynamics and oxygen transfer in a 7 L bioreactor with different impellers combinations were simulated by computational fluid dynamics (CFD) model. Based on the simulation results, two impeller combinations with higher oxygen transfer rate (KLa) were selected to conduct CPC fermentations, aiming at achieving high CPC concentration and low accumulation of major by-product, deacetoxycephalosporin (DAOC). It was found that an impeller combination with a higher KLa and moderate shear force is the prerequisite for efficient CPC production in a stirred bioreactor. The best impeller combination, which installed a six-bladed turbine and a four-pitched-blade turbine at bottom and upper layers but with a shortened impellers inter-distance, produced the highest CPC concentration of 35.77 g/L and lowest DAOC/ CPC ratio of 0.5%.