In Arabidopsis, the MYC2-family basic helix-loop-helix transcription factors mediate transcriptional regulation of jasmonate-responsive genes, and their transcriptional activities are suppressed by physical interactions with jasmonate-ZIM domain (JAZ) proteins. Jasmonate-inducible nicotine formation in Nicotiana plants has been shown to be suppressed by tobacco JAZ proteins, and be regulated by both MYC2-related and NIC2-locus ethylene response factor (ERF) transcription factors. We here show that tobacco MYC2 (NtMYC2) recognizes the G-box sequences, 5'-CAC(G/A)T(G/T)-3', found in the proximal promoter regions of several nicotine biosynthesis genes, including Putrescine N-Methyltransferase 2 (PMT2) and Quinolinate Phosphoribosyltransferase 2 (QPT2). Transient transactivation assays using cultured tobacco cells showed that NtMYC2 and NIC2-locus ERF189 additively activated the PMT2 and QPT2 promoters depending on their cognate binding sites. RNA interference (RNAi) silencing of NtMYC2 in tobacco hairy roots strongly decreased transcript levels of jasmonate-responsive structural genes, including those involved in nicotine biosynthesis, as well as the NIC2-locus ERF genes. Conversely, ERF189 was not required for the expression of NtMYC2. NtMYC2, but not ERF189, interacted with tobacoo JAZs in a yeast two-hybrid assay. These results indicate that NtMYC2 controls nicotine biosynthesis genes in two combinatorial ways, by directly binding the G-box in the target promoters and by up-regulating the NIC2-locus ERF genes.