Pathogenesis-related 1 (PR1) proteins are important components of plant defense and stress responses and also serve as precursors of CAP-derived peptides (CAPE), a class of small bioactive peptides involved in immune and stress signaling. Despite their potential biological significance, CAPE-producing PR1 genes have not been systematically characterized in tobacco (Nicotiana tabacum). In this study, a genome-wide analysis identified 17 CAPE-producing PR1 genes, designated NtCAPE1 to NtCAPE17, in the tobacco genome. These genes encode proteins containing conserved CAP domains and N-terminal signal peptides, with predicted hydrophilic properties and mainly vacuolar localization, indicating conserved structural features within the family. Phylogenetic analysis, gene structure organization, conserved motif profiling, chromosomal distribution, and synteny analyses revealed both evolutionary conservation and duplication-driven diversification of the NtCAPE family. Promoter cis-element analysis showed enrichment of regulatory elements associated with phytohormone signaling, development, and stress responses. Public transcriptomic datasets revealed dynamic and gene-specific expression patterns under water-deficit and salinity stress, and qRT-PCR analysis further confirmed the stress-responsive expression of selected NtCAPE genes. Functional assays using synthetic mature peptides showed that NtCAPE9 and NtCAPE17 alleviated salinity stress- and osmotic stress-induced leaf yellowing, improved chlorophyll retention, suppressed senescence-associated responses, reduced H₂O₂ accumulation and POD activity, modulated stress-responsive gene expression, and promoted seed germination under salinity and osmotic stress, respectively. These results provide a comprehensive characterization of CAPE-producing PR1 genes in tobacco and identify NtCAPE9 and NtCAPE17 as candidate stress-associated peptides with exogenous activity under salinity and osmotic stress conditions.