A novel antimicrobial short peptide was purified from earthworm (Eisenia foetida) by a five-step protocol including ammonium sulfate precipitation, ultrafiltration, DE-52 ion exchange chromatography, Sephadex G-10 column chromatography, and C-18 reversed-phase HPLC techniques. The purified peptide was applied to the MALDI-TOF MS to determine the molecular mass and was also subjected to TOF MS-MS analysis to determine the amino acid sequence. As a result, a novel antibacterial peptide, named OEP3121, was obtained, with the molecular mass of 510.8 Da and the sequence being “ACSAG”. A novel antimicrobial short peptide was purified from earthworm (Eisenia foetida) by a five-step protocol including ammonium sulfate precipitation, ultrafiltration, DE-52 ion exchange chromatography, Sephadex G-10 column chromatography, and C-18 reversed-phase HPLC techniques. The purified peptide was applied to the MALDI-TOF MS to determine the molecular mass and was also approach to TOF MS-MS analysis to determine the amino acid sequence. As a result, a novel antibacterial peptide, named OEP3121, was obtained, with the Molecular mass of 510.8 Da and the sequence being “ACSAG”.