Abstract In plant, some WRKY transcription factors are known to play an important role in the transcriptional reprogramming associated with the immune response. By using WRKY-domain-specific differential display procedure, we isolated CaWRKYb gene, which is rapidly induced during an incompatible interaction between hot pepper and Tobacco mosaic virus (TMV) pathotype P 0 infection. The recombinant CaWRKYb bound to the W box-containing CaPR-10 promoter probes efficiently and the specificity of binding was confirmed by mutant study and competition with cold oligonucleotides. Also, in GUS reporter activity assay using Arabidopsis protoplasts with the CaPR-10 promoter, GUS activity was increased in the presence of CaWRKYb. And CaWRKYb-knockdown plant showed reduced number of hypersensitive response local lesions upon TMV-P 0 infection. Furthermore, CaWRKYb-knockdown plant exhibited compromised resistance to TMV-P 0 by accumulating more TMV, apparently through decreased expression of CaPR-10, CaPR-1, and CaPR-5. These results suggest that CaWRKYb is involved as a positive transcription factor in defense-related signal transduction pathways in hot pepper.