Abstract: To elucidate the role of C2H2-type zinc finger proteins (C2H2-ZFP) in tobacco nicotine synthesis, 131 transcription factor family members of NtC2H2-ZFP were identified from the genome of common tobacco K326 via genome-wide identification. Phylogenetic analysis classified these members into 6 subfamilies. Promoter and transcriptome analyses revealed that NtC2H2-ZFP family members exhibited distinct expression patterns characterized by significant tissue specificity and stress responsiveness, and their promoter regions were enriched in light- and hormone-responsive elements. Through further integration of expression pattern and co-expression analyses, NtZFP26 was selected as a candidate gene associated with nicotine synthesis. It showed specific high expression in the root cortex—the key site of nicotine synthesis—and was significantly induced by topping and methyl jasmonate (MeJA). Moreover, NtZFP26 was co-expressed with key nicotine biosynthesis genes, including NtPMT2, NtPMT3, NtPMT4 and NtBBL. Functional verification demonstrated that the ntzfp26 mutant generated by CRISPR/Cas9 technology showed a marked decrease in nicotine content in both roots and leaves compared with the wild type, accompanied by downregulated expression of key nicotine synthesis genes. Yeast one‑hybrid and dual‑luciferase reporter assays confirmed that NtZFP26 directly binds to the promoter of the rate‑limiting enzyme gene NtQPT2 and activates its transcription. In summary, NtZFP26 acts as a key positive regulator in the tobacco nicotine biosynthesis pathway, and regulates nicotine accumulation by activating NtQPT2 expression.