It is mandatory to assess the allergenic potential of genetically modified (GM) crops before their commercialization. Recently, a transgene [Calcineurin B-like (CBL) protein] has been introduced into tobacco plant to make the crop salt resistance. Therefore, it was felt necessary to assess the allergenic potential of the cbl gene product, which was introduced and expressed in Nicotiana tabacum (tobacco) plant and compared the allergenic effects with the wild-type (WT) counterpart. Bioinformatic analysis revealed that there was no significant sequence homology with known allergens. Also, no difference between the protein digestibility profiles of GM and WT tobacco was found. Rapid digestion of CBL protein (Mol Wt 35 kDa) by simulated gastric fluid (SGF) indicated reduced chances of this protein to induce allergenicity. In addition, BALB/c mice sensitized by intraperitoneal administration of WT and GM tobacco protein showed comparable levels of clinical score, specific IgE, IgG1, histamine level, similar effect on different organs as well as IgE binding proteins. These findings indicate that insertion of cbl gene in tobacco did not cause any additional allergic risk to consumer and the GM and native tobacco proteins behave similarly in both in vitro and in vivo situations even after genetic modification.