Metagenomic cosmid libraries containing 1.26×105 clones, covering about 4.8×106 kb metagenomic DNA of uncultured microorganisms from the contents of buffalo rumens were constructed, and 118 independent clones expressing b-glucosidase activity were isolated from the libraries. Screening of these clones showed that eight clones expressed relatively higher b-glucosidase activity at pH 5.0 and 37℃. One out of the eight clones was subcloned. Sequencing analysis showed that an open reading frame (ORF) of 2223 bp, termed umcel3G, potentially encodes a b-glucosidase. The encoded product shared highest homology with a b-glucosidase from Bacillus sp. at 60% identity and 73% similarity. The umcel3G was over-expressed in Escherichia coli and the size of the translated product Umcel3G on SDS-PAGE was in agreement with the predicted molecular mass. Zymogram analysis showed that Umcel3G exhibited b-glucosidase activity, confirming that this ORF encodes a b-glucosidase. The Umcel3G, purified with Ni-NTA column, exhibited optimal activity at pH 6.0~6.5 and 45oC. Certain ions such as Ca2+, Zn2+ had significant positive effect on the activity of Umcel3G. However, some ions such as Fe3+, Cu2+ gave significant inhibitory effect on the enzyme. The Ni-NTA purified recombinant b-glucosidase Umcel3G had a specific activity of 22.8 IU/mg at pH4.5, 35oC and at the presence of 5 mmol/L Ca2+, indicating that this enzyme has potential applications in the fermentative production of ethanol by simultaneous saccharification and cofermentation (SSCF) of lignocelluloses.