The 8-hydroxyisocoumarin, named Centipedin, which has a significant antibiotic activity, was separated and solubilized with organic solvents, such as diethyl ether from centipede Scolopendra subspinipes multilans L. Koch. The Centipedin was purified by silicic acid column and high S cation exchange chromatography followed by reverse-phase HPLC. It was confirmed that Centipedin has a potent antibiotic effectiveness against Gram-negative Klebsiella pneumoniae ATCC 8308. The results showed that Centipedin blocks both DNA replication and RNA transcription during the growth of this pathogen in vivo. The biosynthesis of antibiotic 8-hydroxyisocoumarin was studied in vivo by feeding $[^{14}C]-labelled$ compound as a precursor to live centipede, in which $[^{14}C]acetate$ was the most efficiently incorporated into the Centipedin within 30 h after injection. Also, in vitro study on the biosynthesis of Centipedin showed that efficient incorporation of $[^{14}C]acetate$ occurred at pH range 5.0-7.0 for 10 h incubation and decreased significantly after then. It is suggested that 8-hydroxyisocoumarin is one of the defense compounds acting on bacterial infection in Scolopendra subspinipes.