Mitogen-activated protein kinase (MAPK) cascades are important signaling modules in eukaryotic cells that convert signals generated from the receptors/sensors to cellular responses. Upon activation, MAPKs can be translocated into nuclei where they phosphorylate transcription factors, which in turn activate gene expression. We recently identified NtMEK2, a tobacco MAPK kinase, as the upstream kinase of SIPK and WIPK, two well-characterized tobacco stress-responsive MAPKs. In the conditional gain-of-function NtMEK2DD transgenic tobacco plants, the activation of endogenous SIPK and WIPK by NtMEK2DD induces several groups of defense genes, including 3-hydroxy-3-methlyglutaryl CoA reductase (HMGR), basic pathogenesis related (PR) genes, systemic acquired resistance gene 8.2 (Sar 8.2), and harpin-induced gene1 (Hin1). To identify the transcription factor(s) involved in the activation of these defense genes, we performed gel-mobility shift assays using nuclear extracts from NtMEK2DD plants. Among the common cis-acting elements present in the promoters of defense-related genes, we observed a strong increase in the binding activity to the W box in nuclear extracts from the NtMEK2DD plants but not the control NtMEK2KR plants. The elevated W-box-binding activity in the nuclear extracts cannot be reversed by phosphatase treatment, excluding the possibility of a direct phosphorylation regulation of WRKY transcription factors by SIPK/WIPK. Instead, we observed a rapid increase in the expression of several WRKY genes in the NtMEK2DD plants. These results suggest that the increase in W-box-binding activity after SIPK/WIPK activation is a result of WRKY gene activation, and the NtMEK2-SIPK/WIPK cascade is involved in regulating the expression of genes ranging from transcription factors to defense genes further downstream during plant defense responses.