Cre-LoxP homologous recombinant system was used to disrupt eag gene in B. anthracis AP422. To construct the recombinant vector, homologous regions and Spc^r cassette with two loxP sites were amplified from the corresponding templates. The produced shuttle vector was then transformed into B. anthracis AP422. Under the pressure of temperature and antibiotic, homologous recombination occurred between the vector and genome of the host bacterium and the recombinants were selected by agar media with spectinomycin (Spc) and X-gal. To remove the Spc^r cassette, a plasmid with Cre recombinase was introduced into the recombinant to delete the relevant DNA fragment, resulting in a single loxP site within the targeted genomic segment. The markerless mutant strains were detected by genome PCR, RT-PCR, total proteins SDS-PAGE analysis and Western blot. The results showed that the eag gene was successfully deleted.