To study the toxin-antitoxin system mazEF6 of Mycobacterium tuberculosis (M. tuberculosis), deletion mutants were constructed and subjected to phenotype analysis. First, the flanking (homology arms) of mazEF6 gene from H37Rv and kanamycin resistance gene (kan gene) from plasmid PUC-19K were amplified by polymerase chain reaction (PCR) respectively. Second, fusion PCR was used for the hybrid splicing of mazEF6 homology arms and kan gene, and the desired fusion fragment was obtained. Then the fragment was cloned into pMD-19T (simple) vector to form a suicide plasmid (pMD-19T-ΔmazEF6-kan), and the suicide plasmid was transformed into Escherichia coli (E. coli) DH5α. At last, the constructed plasmid was transformed into H37Rv by electroporation. Single colonies of M. tuberculosis were screened on L-J medium with kanamycin, the genomic DNA of positive strains were extracted, and the targeted fragments were amplified by PCR and sequenced. The genetic stability and other phenotypes of the H37Rv ΔmazEF6 deletion mutant were studied. The results showed that the deletion mutant strains did not present reverse mutant within 15 generations. Compared with the wild-type strains, H37Rv ΔmazEF6 deletion mutant strains grew more slowly and the bacterial cell was relatively shorter. This study demonstrated that it is practical to obtain M. tuberculosis deletion mutant by fusion PCR technology, and the survival ability of M. tuberculosis without toxin-antitoxin mazEF6 gene is decreased.