The voltage-gated calcium channel (VGCC) β subunit (Cavβ) protein is a kind of cytosolic auxiliary subunit that plays an important role in regulating the surface expression and gating characteristics of high-voltage-activated (HVA) calcium channels. Ditylenchus destructor is an important plant-parasitic nematode. In the present study, the putative Cavβ subunit gene of D. destructor, namely, DdCavβ, was subjected to molecular characterization. In situ hybridization assays showed that DdCavβ was expressed in all nematode tissues. Transcriptional analyses showed that DdCavβ was expressed during each developmental stage of D. destructor, and the highest expression level was recorded in the third-stage juveniles. The crucial role of DdCavβ was verified by dsRNA soaking-mediated RNA interference (RNAi). Silencing of DdCavβ or HVA Cavα1 alone and co-silencing of the DdCavβ and HVA Cavα1 genes resulted in defective locomotion, stylet thrusting, chemotaxis, protein secretion and reproduction in D. destructor. Co-silencing of the HVA Cavα1 and Cavβ subunits showed stronger interference effects than single-gene silencing. This study provides insights for further study of VGCCs in plant-parasitic nematodes.