AcGH30A and AcXyn30B_12 are two of the most abundant enzymes in the cellulosome of the thermophilic anaerobe Acetivibrio clariflavus. Their surprising abundance within the glycolytic repertoire of this highly efficient microorganism, active in sewage sludge ecosystems, suggests a cooperative role in the hydrolysis of complex carbohydrates. Here, we cloned, expressed and characterized the endo-β-1,4-xylanase AcXyn30B_12, which has a molecular weight of ~74 kDa and displays optimal activity at pH 5.5 and 70 °C. AcXyn30B_12 exhibited broad substrate specificity, with the highest catalytic efficiency against partially acetylated birchwood xylan (PABX), yielding a V