A spermine-based signal transduction pathway plays a defensive role against incompatible pathogens. We identified a novel spermine-responsive cDNA from Nicotiana tabacum that encodes a basic region/leucine zipper protein with a putative transmembrane domain. Identity to Arabidopsis thaliana AtbZIP60 was sufficiently high to name the novel cDNA NtbZIP60. Expression analysis revealed that NtbZIP60 is a component of the spermine-signal pathway, and is also involved in the unfolded protein response (UPR), as demonstrated for AtbZIP60. The gene product, NtbZIP60, localizes to the endoplasmic reticulum (ER) in plant cells; once the putative transmembrane domain is eliminated from the intact protein, it targets the nucleus. The putative processed form of NtbZIP60 transactivates target genes through binding to plant-specific UPR cis-elements. Expression of NbbZIP60, an NtbZIP60 ortholog in Nicotiana benthamiana, was significantly up-regulated at 6 h and later time points upon infection with the non-host pathogen Pseudomonas cichorii, while it was unaffected by infection with the compatible pathogen Pseudomonas syringae pv. tabaci. Furthermore, NbbZIP60-silenced N. benthamiana plants allowed higher multiplication of P. cichorii compared to the control plants. Taken together, the results suggest that this ER-localized transcription factor is involved in the spermine-signal transduction pathway and plays an important role in plant innate immunity.