We examined butanol fermentation by Clostridium beijerinckii NCIMB 8052 using various hydrolyzates obtained from rice bran, which is one of the most abundant agricultural by-products in Korea and Japan. In order to increase the amount of fermentable sugars in the hydrolyzates of rice bran, various hydrolysis procedures were applied. Eight different hydrolyzates were prepared using rice bran (RB) and defatted rice bran (DRB) with enzyme or acid treatment or both. Each hydrolyzate was evaluated in terms of total sugar concentration and butanol production after fermentation by C. beijerinckii NCIMB 8052. Acid treatment yielded more sugar than enzyme treatment, and combined treatment with enzyme and acid yielded even more sugars as compared with single treatment with enzyme or acid. As a result, the highest sugar concentration (33 g/l) was observed from the hydrolyzate from DRB (100 g/l) with combined treatment using enzyme and acid. Prior to fermentation of the hydrolyzates, we examined the effect of P2 solution containing yeast extract, buffer, minerals, and vitamins on production of butanol during the fermentation. Fermentation of the hydrolyzates with or without addition of P2 was performed using C. beijerinckii NCIMB 8052 in a 1-1 anaerobic bioreactor. Although the RB hydrolyzates were able to support growth and butanol production, addition of P2 solution into the hydrolyzates significantly improved cell growth and butanol production. The highest butanol production (12.24 g/l) was observed from the hydrolyzate of DRB with acid and enzyme treatment after supplementation of P2 solution.