Entomopathogenic nematodes are being used for insect control. We purified a toxin secreted by the insect-pathogenic bacterium,Xenorhabdus nematophilus, which lives in the gut of entomopathogenic nematodes. Culture broth ofX. nematophilus was separated by centrifugation and concentrated by ultrafiltration. The concentrated culture broth was applied to a DEAE Sephadex A-50 column, and proteins were eluted stepwise with increasing concentrations of KCl. Fractions with insect toxicity were further concentrated and then applied to a HPLC with a gel filtration column. The molecular weight of purified toxin was 39 kDa on SDS-PAGE, and Fourier transformed infrared (FTIR) spectroscopy indicated that this toxin could be a new protein exhibiting the characteristics of C=O stretching peak near 1650 cm−1.