The gene encoding an esterase enzyme was cloned from a metagenomic library of cow rumen bacteria. The esterase gene (est5S) was 1,026 bp in length, encoding a protein of 366 amino acid residues with a calculated molecular mass of 40,168 Da. The molecular mass of the enzyme was estimated to be 40,000 Da. The Est5S protein contains the Gly-X-Ser-X-Gly motif found in most bacterial and eukaryotic serine hydrolases. However, the Asp or Glu necessary for the catalytic triad [Ser-Asp-(Glu)-His] was not present, indicating Est5S represents a novel member of the GHSQG family of esterolytic enzymes. BlastP in the NCBI database analysis of Est5S revealed homology to hypothetical proteins and it had no homology to previous known lipases and esterases. Est5S was optimally active at pH 7.0 and $40^{circ}C$. Among the p-nitrophenyl acylesters tested, high enzymatic activities were observed on the short-chain p-nitrophenyl acylesters, such as p-nitrophenyl acetate, etc. The conserved serine residue ($Ser_{190}$) was shown to be important for Est5S activity. The primers that amplified the est5S gene did not show any relative band with 49 species of culturable rumen bacteria. This implies that a new group esterase gene, est5S, may have come from a noncultured cow rumen bacterium.