Bursaphelenchus xylophilus is the pathogen, which causes pine wilt disease (PWD). The disease has caused significant economic losses and damage to forests. However, the pathogenic mechanism of B. xylophilus remains unclear. Cystatins are involved in various biological processes where they regulate normal proteolysis and also play a role in pathogenicity, but their functions in B. xylophilus are unknown. Therefore, we cloned the full-length cDNA of a cystatin gene of B. xylophilus (Bx-cpi-1) by rapid-amplification of cDNA ends and analyzed its characteristics with bioinformatic methods. In situ mRNA hybridization analyses showed that transcripts of Bx-cpi-1 were abundantly expressed in the reproductive organs of B. xylophilus. The expression of Bx-cpi-1 was investigated using qPCR. Bx-cpi-1 was expressed during each of the different developmental stages of B. xylophilus. The highest gene expression was at the egg stage. After infection of Pinus massoniana, the expression of Bx-cpi-1 increased. The functions of Bx-cpi-1 were verified by RNA interference. The feeding rate, reproduction and pathogenicity of B. xylophilus all decreased as a result of silencing of the Bx-cpi-1 gene. These results revealed that Bx-cpi-1 may be a variant of a type II cystatin gene which is involved in the development and pathogenic process of B. xylophilus.