Objective To clone the cDNA sequence of Attacin gene from Musca domestica larvae, construct prokaryotic fusion expression vectors, express and purify the Attacin protein, and preliminarily study its antibiotic biological function. Methods The cDNA fragment encoding Attacin was amplified from recombinant plasmid pUCm-T/Attacin by PCR with specific primers and then cloned into pET30a (a + ) and pGEX-4T-1 vector separately. These prokaryotic expression recombinant plasmids were transformed into E. coli to express the relative proteins respectively. The antibacterial activity of Attacin in E. coli was monitored with an in vivo system. The expressed recombinant products were purified with affinity-chromatograph column and analyzed with SDS-PAGE. Then the biological activities of Attacin were detected by agarose plate antibiotic assay. Results The 674 bp cDNA of Attacin was obtained by RT-PCR. The sequence has been accepted by GenBank (Accession number DQ062744). The pET30a (a + ) /Attacin and pGEX-4T-1/Attacin recombinant plasmids were confirmed by PCR, restriction endonuclease digestion and DNA sequencing, respectively. Transformation assay of E. coli with the recombinant plasmids revealed that, after IPTG induction and compared to non-induced control, the growth of host cells containing pET30a ( + ) / Attacin was significantly suppressed, with no His-Attacin band on SDA-PAGE as expected. Although the growth of host cells containing pGEX-4T-1/Attacin was also inhibited, the GST-Attacin fusion protein was obtained. The GST-Attacin fusion protein then underwent GST affinity-chromatography, and SDS-PAGE analysis confirmed the final purified product had the expected size. The recombinant protein exhibited antibacterial activities when assayed with agarose plates inoculated with bacteria. Conclusion The Attacin expressed in prokaryotic system possesses antibacterial activities, which lays a reliable foundation for further research on biological functions and application of Attaicn.