From a biogas reactor metagenome an ORF (bpcel9A) encoding a bacterial theme C glycoside hydrolase family 9 (GH9) enzyme was recombinantly produced in E. coli BL21 pQE-80L. BPCel9A exhibited ≤ 55% identity to annotated sequences. Subsequently, the enzyme was purified to homogeneity by affinity chromatography. The endo-beta-glucanase BPCel9A hydrolyzed the beta-1,3–1,4-linked barley beta-glucan with 24 U/mg at 30 °C and pH 6.0. More than 62% of activity was measured between 10 and 40 °C. Lichenan and xyloglucan were hydrolyzed with 67% and 40% of activity, respectively. The activity towards different substrates varied with different temperatures. However, the enzyme activity on CMC was extremely low (> 1%). In contrast to BPCel9A, most GH9 glucanases act preferably on crystalline or soluble cellulose with only side activities towards related substrates. The addition of calcium or magnesium enhanced the activity of BPCel9A, especially at higher temperatures. EDTA inhibited the enzyme, whereas EGTA had no effect, suggesting that Mg2+ may adopt the function of Ca2+. BPCel9A exhibited a unique substrate spectrum when compared to other GH9 enzymes with great potential for mixed-linked glucan or xyloglucan degrading processes at moderate temperatures.