Anthrax toxin is one of the two virulence factors of Bacillus anthracis and it is a binary bacterium toxin composed of protective antigen (PA), lethal factor (LF) and edema toxin (EF). Among of them, LF is the enzyme moiety lead to the death of the cell. To investigate key function amino acid of LF and study the lethal mechanism of lethal factor, random mutagenesis was carried out. The mutation library of the 774 bp fragment of lethal factor from site 1 550 to 2 324 bp was constructed using Diversify^TM PCR Random Mutagenesis Kit (Clontech) with some modifiication. Transformants of lef mutation library were induced by 0.2 mmol/L IPTG at 28℃ for 4 h and then T7 phage were added into the culture, incubating for further 2～3 h to lyse the bacterium. The lysis was centrifuged at 12 000 r/min for 8 min and the supernatant which contained the target protein was collected. To determine the activity of the mutants, the cell cytotoxicity assay was carried out. RAW 264.7 microphage cells were seeded into 96-well plates, 24 h ahead of experiments. When assaying, the medium was removed and 100 μl DMEM medium containing 1.0 mg/L of purified recombinant and 10 μl of the lysis supernatant was added into each well. Then the plates were incubated at 37℃ for 4 h. The cell viability was determined by using the alamarBlueTM (AbD Serotec) dye according to the manufacturer’s direction. The mutants whose activities had decreased significantly were sequenced and further purified by Ni-histidine tag affinity chromatography. The cell cytotoxicity and competition binding assay were demonstrated to analyse the character of the mutants. Five mutants which had low biological activity determined through the cell cytotoxicity assay were obtained. Furthermore, two mutant proteins with single-site mutation, K518E and L519C, were purified for the first time. The cytotoxicity assay showed that the two mutant proteins lost activity significantly, that means, Lys518 and Leu519 played an important role in the function of lethal factor. The competition binding assay showed K518E endowed with the character of competition inhibition of wild type LF, whereas L519C doesn’t have the character. Probably, mutant K518E change Lys into Glu carrying negative charge and affects the catalytic groove of LF which is negative charge. Therefore, Lys 518 maybe has an important role to stabilize the conformation of LF and combine the substrate.