As a lignocellulose-based substrate for anaerobic digestion, rice straw is characterized by low density, high water absorbability, and poor fluidity. Its mixing performances in digestion are completely different from traditional substrates such as animal manures. Computational fluid dynamics (CFD) simulation was employed to investigate mixing performances and determine suitable stirring parameters for efficient biogas production from rice straw. The results from CFD simulation were applied in the anaerobic digestion tests to further investigate their reliability. The results indicated that the mixing performances could be improved by triple impellers with pitched blade, and complete mixing was easily achieved at the stirring rate of 80 rpm, as compared to 20-60 rpm. However, mixing could not be significantly improved when the stirring rate was further increased from 80 to 160 rpm. The simulation results agreed well with the experimental results. The determined mixing parameters could achieve the highest biogas yield of 370 mL (g TS)(-1) (729 mL (g TSdigested)(-1)) and 431 mL (g TS)(-1) (632 mL (g TSdigested)(-1)) with the shortest technical digestion time (T (80)) of 46 days. The results obtained in this work could provide useful guides for the design and operation of biogas plants using rice straw as substrates.