The evolutionarily conserved mitogen-activated protein kinase (MAPK) cascades relay extracellular signals into cells, triggering a variety of cellular responses. We previously revealed NtMPK4 as a positive regulator of nicotine biosynthesis; however, its upstream regulation remains unclear. Here, we characterized a MAPK cascade, comprising NtMEKK1b, NtMPKK2a, and NtMPK4, that promotes nicotine biosynthesis. This signaling module transduces external cues, including jasmonate and pathogen elicitors such as flg22, into post-translational modifications that enhance transcriptional activity and pathway gene expression. NtMPKK2a physically interacts with and phosphorylates NtMPK4 in vivo, confirming its role as an upstream kinase. RNAi-mediated silencing of NtMPKK2a significantly reduced the expression of nicotine pathway genes and decreased nicotine accumulation, whereas induced-overexpression of NtMPKK2a upregulated nicotine pathway genes and increased nicotine contents in tobacco hairy roots. Overexpression of NtMPKK2a in tobacco cells enhanced the transactivation activity of a NIC2-locus Ethylene Response Factor NtERF221 on Putrescine N-methyltransferase (NtPMT) promotor, further supporting its role in promoting nicotine biosynthesis. Furthermore, we identified NtMEKK1b, a tobacco MEKK that interacts with NtMAPKK2a in yeast cells. Knock-down of NtMEKK1b in transgenic tobacco plants attenuated the expression of nicotine pathway genes and reduced nicotine contents, whereas induced-overexpression of NtMEKK1b upregulated gene expression and nicotine accumulation. Our findings uncover a previously uncharacterized MAPK cascade module, NtMEKK1b-NtMPKK2a-NtMPK4, that regulates nicotine biosynthesis, highlighting the importance of posttranslational regulation in nicotine biosynthesis.