The plant proteinase inhibitor-II (PI-II) proteins are diverse group of proteins which have been intensively investigated due to their potential role in providing plant protection against different environmental stresses. These genes have been implicated in the regulation of various physiological functions in plants such as modulation of plant growth and development, regulation of endogenous proteases, and mediating defense responses. In the present study, PI-II gene sequences from ten different plants were computationally analyzed using bioinformatics tools. The obtained results identified highly conserved domains in all analyzed sequences. Further, phylogenetic comparisons of PI-II genes representing ten different plants suggested that the high rate of retention of gene duplication and inhibitory domain multiplication may have resulted in the expansion and functional diversification of these proteins. The induction of PI-II gene is stimulated by various physical and chemical signals like mechanical wounding and by interplay of related phytohormones. Therefore, Agrobactreium-mediated transformation was carried out in tobacco with tomato PI-II gene under the regulation of rice root germin-like protein 2 (OsRGLP2) promoter. T1 transgenic seedlings were used for expression analysis in response to wounding, abscisic acid (ABA), methyl jasmonate (MeJA) and salt stress treatments. From the results of qPCR, it was found that ABA and MeJA dependent signaling pathways are involved in stimulation of PI-II gene. The PI-II induction by ABA and MeJA indirectly indicates its defensive role against environmental biotic and abiotic stresses such as insects, pathogen, drought and salt stresses. Transgenic seedlings subjected to salt stress condition showed significant PI-II gene activity under OsRGLP2 promoter in transgenic lines. Phenotypic analysis revealed that transgenic plants had comparatively higher level of chlorophyll content, proline content and average fresh weight than wild type plants under salt stress. Moreover, relatively high GUS expression was detected in the vascular regions of leaves, stem and roots after treatment with ABA, MeJA, wounding and salt stresses. The findings of present research are useful for enlightening the role of OsRGLP2 driven PI-II gene expression to improve stress tolerance in transgenic crops.