Bursaphelenchus xylophilus (PWN) causes pine wilt disease (PWD), which is one of the most devastating pine diseases worldwide. Cytochrome P450 (CYP) catalyzes the biosynthetic metabolism of terpenoids and plays an important role in the modification of secondary metabolites in all living organisms. We investigated the molecular characteristics and biological functions of Bx-cyp29A3 in B. xylophilus. The bioinformatics analysis results indicated that Bx-cyp29A3 has a transmembrane domain and could dock with L(−)-carvone. The gene expression pattern indicated that Bx-cyp29A3 was expressed in 0.2, 0.4, 0.6, 0.8, and 1.0 mg/mL L(−)-carvone solutions. The Bx-cyp29A3 expression increased in a dose-dependent manner and peaked at 24 h of exposure when the L(−)-carvone solution concentration was 0.8 mg/mL. However, the gene expression peaked at 0.6 mg/mL after 36 h. Furthermore, RNA interference (RNAi) indicated that Bx-cyp29A3 played an essential role in the response to L(−)-carvone. The mortality rates of the Bx-cyp29A3 knockdown groups were higher than those of the control groups in the 0.4, 0.6, 0.8, and 1.0 mg/mL carvone solutions after 24 h of exposure or 36 h of exposure. In summary, bioinformatics provided the structural characteristics and conserved sequence properties of Bx-cyp29A3 and its encoded protein, which provided a target gene for the study of the P450 family of B. xylophilus. Gene silencing experiments clarified the function of Bx-cyp29A3 in the immune defense of B. xylophilus. This study provides a basis for the screening of new molecular targets for the prevention and management of B. xylophilus.