A cellular signal transduction pathway induced by the polyamine, spermine (Spm), and transmitted by mitochondrial dysfunction is proposed in tobacco. In this investigation, we further resolve the pathway by identifying a subset of hypersensitive response (HR) marker genes as downstream components. In a previous report, we identified harpin‐induced 1 (HIN1) and two closely related genes as responsive to Spm. Other HR marker genes, HSR203J, HMGR, HSR201, and HSR515, are also Spm‐responsive. Induction of these HR marker genes, including HIN1, by Spm was suppressed by pre‐treatment with antioxidants, calcium channel blockers, inhibitor of mitochondrial permeability transition pore openings, and blockers of amine oxidase/polyamine oxidase. Such quenching is also observed for Spm‐induced activation of two mitogen‐activated protein kinases (MAPKs), salicylic acid‐induced protein kinase (SIPK), and wound‐induced protein kinase (WIPK), and upregulation of the WIPK gene, suggesting that all these components are part of the same signaling pathway. Furthermore, gain‐of‐function and loss‐of‐function studies on MAPK cascade members reveal that the expression of Spm‐induced HR marker genes varies with respect to involvement of SIPK/WIPK activation.