To discover and isolate a glyphosate-resistant gene from a microorganism through gene mining. The full aroM gene from Acremonium sp. (named aroM (A.sp.)) was cloned using rapid amplification of cDNA ends. The transcriptional expression level of each domain increased significantly after glyphosate treatment in the aroM (A.sp.) complex and reached its maximum at 48 h. The aroA domain of the aroM (A.sp.) (named aroA (A.sp.)) was expressed in Escherichia coli BL21 (DE3) and the product was purified through Ni-NTA affinity chromatography. Furthermore, 45 KDa was indicated by SDS-PAGE and its enzyme activity was optimal at 30 A degrees C and PH 7.0. The K-i/K-m value of aroA (A.sp.) was 0.106, and the E. coli BL21 harboring aroA (A.sp.) could grow in the M9 minimal medium with 100 mM glyphosate. The aroA (A.sp.) from the aroM (A.sp.) complex had high enzyme activity and glyphosate resistance. Therefore, this research offers a new strategy for improving glyphosate resistance using the aroA domain of the aroM complex in the fungi.