A combination of rice straw and Bacillus subtilis (B. subtilis) was used to remove methylene blue (MB) from wastewater at varied operational conditions. Virtually identical reductions in MB of 58.4, 61.8, 64.6, and 64.6mg from 1L solution were achieved for different initial MB concentrations (100, 200, 300, and 400mg/L) when rice straw was used alone, which were increased to 91.6, 184.6, 275.3, and 368.1mg after the rice straw was inoculated with B. subtilis. The amounts of total removed MB in the treatment groups were almost 1.5, 3, 4.5, and 6 times of that in the control group without B. subtilis. These results demonstrated that the combination of rice straw and B. subtilis was effective for the removal of MB from aqueous solutions. In the four treatment groups, of the total removed MB, 52.5%, 58.7%, 66.4%, and 76.3% were adsorbed by rice straw and bacterial biomass, respectively, and the rest were removed by microbial biodegradation. The significant improvement in MB removal was mainly because the B. subtilis increased the specific surface area of the straw and produced more functional groups on it. Additionally, microbial biodegradation was another reason for the improvement in MB removal. Furthermore, inoculation of rice straw with B. subtilis represented an efficient means of removing MB from wastewater in an actual project in Minkang Printing and Dyeing Co., Ltd., Sichuan, China. After 4days of treatment by 2.5g of rice straw (per liter of wastewater) inoculated with B. subtilis, a 94.5% decolorization rate was achieved, which was higher than that of the currently used biological filter with activated carbon as a filter bed (78.3%).