Abstract: Bacillus amyloliquefaciens strain JKJS3 was isolated from the needles of Pinus massoniana, which demonstrated with high nematicidal activity on pine wood nematode Bursaphelenchus xylophilus. The culture conditions of strain JKJS3 were studied in the lab. The results showed there were no significant differences in nematicidal effects on B.xylophilus by metabolic materials secreted from JKJS3 under media with different carbon sources, but the significant differences were observed in those with different nitrogen sources.The best nitrogen sources of the culture media is the mixture of beef extract 5 g/L and peptone 6 g/L. The optimal culture condition for secreting nematicidal materials by JKJS3 is shaking cultured the media(pH=7) at 37 ℃ without light. The obvious correlation between the culture time and the nematicidal activity is observed and the optimal culture time is 10 days. The study may provide information for controlling the pine wood nematode by the strain JKJS3 and its nematicidal materials in future.