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Studying sex communication is necessary to develop new methods to control the population expansion of gonochoristic species Bursaphelenchus xylophilus, the pathogen of pine wilt disease (PWD). Small chemical signals called ascarosides have been reported to attract potential mates. However, they have not been studied in the sex attraction of B. xylophilus. Here, we confirmed the sex attraction of B. xylophilus using a chemotaxis assay. Then, we cloned the downstream ascaroside biosynthetic gene Bx-daf-22 and explored its function in the sex attraction of B. xylophilus through bioinformatics analysis and RNA interference. The secretions of females and males were the sources of sex attraction in B. xylophilus, and the attractiveness of females to males was stronger than that of males to females. Compared with daf-22 of Caenorhabditis elegans, Bx-daf-22 underwent gene duplication events, resulting in Bx-daf-22.1, Bx-daf-22.2, and Bx-daf-22.3. RNA interference revealed that the attractiveness of female secretions to males increased after all three Bx-daf-22 genes or Bx-daf-22.3 had been interfered. However, the reciprocal experiments had no effect on the attractiveness of male secretions to females. Thus, Bx-daf-22 genes, especially Bx-daf-22.3, may be crucial for the effectiveness of female sex attractants. Our studies provide fundamental information to help identify the specific components and signal pathways of sex attractants in B. xylophilus.