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BACKGROUND Research on selenium nanoparticles (SeNPs) in chemical defense and chemotherapy of plants has developed rapidly owing to their high microbial toxicity, environmental safety, and degradability. Pine wilt disease (PWD) threatens pine forests worldwide; however, it is difficult to kill the nematodes (Bursaphelenchus xylophilus) inside the tree that cause PWD using traditional pesticide formulations. SeNPs could be the key to controlling PWD. RESULTS In this study, approximately 50 nm SeNPs were prepared using a simple and green method, and chitosan was used to increase their biocompatibility and stability. The preparation and characterization results showed that the prepared SeNPs coated with chitosan (SeNPs@CS) were spherical and evenly dispersed. The bioassay results showed that SeNPs@CS had an LC50 of 15.627 mg/L against B. xylophilus. In addition, the killing mechanism of SeNPs@CS against B. xylophilus was studied. Confocal microscopy and transmission electron microscopy demonstrated that B. xylophilus were killed by reactive oxygen species, and the penetration of nano-form materials to B. xylophilus was higher than that of non-nano-form materials. To verify the effective penetration of SeNPs in pine tissues, Cy5-labeled SeNPs@CS was observed inside pine needles and branches using frozen sections and confocal microscopy. In addition, the cytotoxicity of SeO2 and SeNPs@CS was tested, and the results showed that the cytotoxicity of SeNPs@CS to MC3T3-E1 cells was reduced. CONCLUSION These results show that SeNPs are expected to be used as a new strategy for the control of PWD with oxidative damage and high penetration to B. xylophilus and effective target penetration and biosafety. This article is protected by copyright. All rights reserved.