Tachyplesin is a 17-aa peptide, isolated from marine “living fossil” horseshoe crab, Tachypleus tridentatus, showing wide-spectrum antibacterial activity. However, its mechnism of killing bacterium is not very clear. The aim was to investigate the targets of tachyplesin for further studying the antibacterial molecular mechnism. In vitro bacterial inhibition method was used to determine feature of bacterial inhibition dynamic of tachyplesin. Inorganic phosphate measurement and ultraviolet absorption methods were used to observe the leakage of inorganic phosphorus and large mass molecular before and after bacterium were incubated with tachyplesin. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were adopted to investigate the morphologic and structural changes of bacterium before and after being incubated with tachyplesin. Ultroviolet absorption method and electrophoretic mobility shift assay (EMSA) were used to investigate the effect of tachyplesin on structure of genomic DNA and plasmid DNA of bacterium. Plasmid transformation method was applied to observe the effect of tachyplesin on copy and transcription of plasmid DNA. The results showed that, (1) Tachyplesin had different feature of bacterial inhibition dynamic in Gram positive and negative bacterium. In the Gram positive bacterium (E. coli K88 and E. coli F41), the antibacterial activity of tachyplesin increased sharply during the first 10 h, and was in the plateau phase during 10～30 h, and then decreased slowly. In the Gram negative bacterium (B. subtilis W B800 and S. aureus), the antibacterial activity of tachyplesin increased sharply during the first 3 h, and decreased during 3～10 h, and increased again during 10～15 h, the trend was similar to the Gram positive bacterium after 20 h. (2) Bacterium lost inorganic phosphorus and large mass molecular. Phosphorous concentration in the culture media of S. aureus was significantly higher after treated with tachyplesin, the high concentration could keep long time (30 h later). The concentration of the UV absorbing substance in E. coli K88 became higher and higher depending on tachyplesin concentration and time. At low tachyplesin concentration, the UV absorbing substance concentration kept low. (3) The structure of cell wall, cell membrane and the whole cell body were damaged to some extent after treated by tachyplesin. (4) Tachyplesin could combine with genomic DNA and plasmid DNA of bacterium, high concentration of tachyplesin probably could break down DNA, and inhibited copy and transcription of plasmid DNA. It was suggested that the antibacterial targets include at least cell wall and membrane and DNA of bacterium.