Abstract: To explore the molecular mechanism of the Bursaphelenchus xylophilus specialized third-stage dauer larva formation, we studied the Bx-DAF6 gene using the RNAi method. Based on the B. xylophilus genome, Bx-DAF6 was identified by the method of BLAST, and cloned by PCR. The gene function was studied by the suppression of Bx-DAF6 transcript in combination with the RNAi method. The results indicated that the Bx-DAF6 CDS was 2 700 bp, which encodes a protein with a domain homologous to sterol-sensing. The suppression of the Bx-DAF6 transcript by RNAi led to a reduction in the number of specialized third-stage dauer larva formation. Our results indicated that Bx-DAF6 regulated positively the formation of specialized third-stage dauer larvae.