De-esterification is an important degradation or detoxification mechanism of sulfonylurea herbicide in microbes and plants. However, the biochemical and molecular mechanisms of sulfonylurea herbicide de-esterification are still unknown. In this study, a novel esterase gene, sulE, responsible for sulfonylurea herbicide de-esterification, was cloned from Hansschlegelia zhihuaiae S113. The gene contained an open reading frame of 1,194 bp, and a putative signal peptide at the N terminal was identified with a predicted cleavage site between Ala37 and Glu38, resulting in a 361-residue mature protein. SulE minus the signal peptide was synthesized in Escherichia coli BL21 and purified to homogeneity. SulE catalyzed the de-esterification of a variety of sulfonylurea herbicides that gave rise to the corresponding herbicidally inactive parent acid and exhibited the highest catalytic efficiency toward thifensulfuron-methyl. SulE was a dimer without the requirement of a cofactor. The activity of the enzyme was completely inhibited by Ag ؉ , Cd 2؉ , Zn 2؉ , methamidophos, and sodium dodecyl sulfate. A sulE-disrupted mutant strain, ⌬sulE, was constructed by insertion mutation. ⌬sulE lost the de-esterification ability and was more sensitive to the herbicides than the wild type of strain S113, suggesting that sulE played a vital role in the sulfonylurea herbicide resistance of the strain. The transfer of sulE into Saccharomyces cerevisiae BY4741 conferred on it the ability to de-esterify sulfonylurea herbicides and increased its resistance to the herbicides. This study has provided an excellent candidate for the mechanistic study of sulfonylurea herbicide metabolism and detoxification through de-esterification, construction of sulfonylurea herbicide-resistant transgenic crops, and bioremediation of sulfonylurea herbicide-contaminated environments. S ulfonylurea herbicides are an important class of herbicides used worldwide for controlling weeds in all major agronomic crops. The herbicides inhibit acetohydroxy acid synthase (AHAS), a key enzyme in the biosynthesis pathway of branched-chain amino acids valine, leucine, and isoleucine in bacteria, fungi, and plants (3,4,8,13). The use of sulfonylurea herbicides has developed rapidly because of their high efficacies at low dosages and multicrop selectivities. The sulfonylurea products are now the second most common kind of herbicides after the glyphosates, and more than 30 products have been commercialized.Most sulfonylurea herbicides are weak acids, vulnerable to acid hydrolysis under acidic conditions. However, in neutral to alkaline soils, some of the herbicides, such as metsulfuron-methyl, chlorsulfuron, and ethametsulfuron-methyl, are degraded at a very slow rate and persist from several months to more than 1 year (15,20,24,30). The residues of herbicides in the soil seriously damage subsequent rotation of sulfonylurea-sensitive crops, like legumes and oilseeds, which can result in serious agricultural loss. Thus, great concern and interest have been raised regarding the enviro...