Anaerobic digestion (AD) of animal manure is traditionally classified as a treatment to reduce the environmental impacts of odor, pathogens, and excess nutrients associated with animal manure. This report shows that AD also changes the composition of manure fiber and makes it suitable as a cellulosic feedstock for ethanol production. Anaerobically digested manure fiber (AD fiber) contains less hemicellulose (11%) and more cellulose (32%) than raw manure, and has better enzymatic digestibility than switchgrass. Using the optimal dilute alkaline pretreatment (2% sodium hydroxide, 130°C, and 2 h), enzymatic hydrolysis of 10% (dry basis) pretreated AD fiber produces 51 g/L glucose at a conversion rate of 90%. The ethanol fermentation on the hydrolysate has a 72% ethanol yield. The results indicate that 120 million dry tons of cattle manure available annually in the U.S. can generate 63 million dry tons of AD fiber that can produce more than 1.67 billion gallons of ethanol. Integrating AD with biorefining will make significant contribution to the cellulosic ethanol production. Biotechnol. Bioeng. 2010;105: 1031–1039. © 2009 Wiley Periodicals, Inc.