A novel esterase (Sf310) from Shewanella frigidimarina, which is composed of 279 amino acids with a molecular mass of 31.0 kDa, was identified, expressed and characterized. This enzyme displayed remarkable identity with S-formylglutathione hydrolases generally known as Esterase D family. Sequence analysis of Sf310 revealed a conserved pentapeptide of G-X-S-X-G found in various lipolytic enzymes and a putative catalytic triad of Ser148-Asp224-His257. Sf310 was active toward short-chain esters such as p-nitrophenyl acetate, butyrate, hexanoate and octanoate, while S148A mutant completely lost its activity. The optimum pH for enzyme activity was slightly alkaline (pH 8.0). Regarding the optimum temperature, interestingly, Sf310 could retain more than 80% of its full activity throughout temperatures ranging from 20 to 120 ℃, although circular dichroism spectroscopy showed that the enzyme is structurally thermolabile above 40 ℃.Moreover, Sf310 displayed above 50% of its initial activity with various chemicals including 30% EtOH, 1% triton x-100, 1% SDS, 5M urea and so on. Taken together, the high thermal and chemical stability of Sf310 could be useful for industrial application and give an insight into the stability of enzymes from Antarctic species.