A recombinant expression plasmid pYH12, containing the double-mutation glucose isomerase (GIG138PG247D, G12) coding gene and its natural regulatory sequence, was constructed for site-specific integration in Streptomyces. The resulting plasmid was introduced into Streptomyces lividans TK54 by protoplast transformation and two apramycin-resistance (Am-R) transformants, designated GY2 and BY7, respectively, were obtained further based on enzyme assays. These results for polymerase chain reaction (PCR), Dot blot, and recovery of cloned fragments from the transformant chromosome indicated that the G12 gene was integrated into the S. lividans chromosome by site-specific recombination, and which was further verified by Southern blot. We found that the free form of plasmid pYH12 co-existing with the integrated form was present in S. lividans. SDS-PAGE analysis showed that the G12 gene was expressed in S. lividans. The intracellular G12 specific activity was 1.15 U/mg. The stability of integrants demonstrated that the cloned G12 gene was stably integrated and expressed even in the absence of selective pressure.