Background: Pine wilt disease (PWD) is a disease caused by the parasitization of Pinus spp. by Bursaphelenchus xylophilus, a pine wood nematode. This nematode is parasitic to insects and is thus transferred to the tree, growing in the tree for a short period of time before blocking nutrients and killing the tree. This is now becoming a serious problem globally. Actinomycetes are known to produce a variety of physiologically active substances. In this study, we search for a substance that kills pine wood nematodes from the secondary metabolites of actinomycetes. Result: We selected Streptomyces sp. AN110065, which has nematicidal activity against B. xylophilus, from among 5,000 actinomycetes. As a result of a 16S rRNA sequence analysis, 99.78% similarity to Streptomyces netropsis NBRC 3723 was found. In addition, by separating the active compound, pimprinethine was identified through an analysis of the 1H-NMR, 13C-NMR, HMQC, 1 H-1H COSY, and HMBC spectra. As a result of evaluating the activity of pimprinethine against B. xylophilus, 100% nematode activity was observed after 24 hours at a concentration of 0.4 mg/ml. Conclusion: This study was performed to isolate useful secondary metabolites produced by soil actinomycetes for the control of pine wilt nematodes. A compound having nematicidal activity was isolated from the selected strain, and its structure was confirmed as pimprinethine. The nematicidal activity of the isolated active ingredient pimprinethine was confirmed, and as the first report on the effect of pimprinethine on PWN, it is proposed as a potential material for the control of pine wood nematodes.