To improve the probability of detecting unintended side effects during maize gene manipulations by bombardment, proteomics was used as an analytical tool complementary to the existing safety assessment techniques. Since seed proteome is highly dynamic, depending on the species variability and environmental influence, we analyzed the proteomic profiles of one transgenic maize variety (event MON 810) in two subsequent generations (T05 and T06) with their respective isogenic controls (WT05 and WT06). Thus, by comparing the proteomic profiles of WT05 with WT06 we could determine the environmental effects, while the comparison between WT06 and T06 seeds from plants grown under controlled conditions enabled us to investigate the effects of DNA manipulation. Finally, by comparison of T05 with T06 seed proteomes, it was possible to get some indications about similarities and differences between the adaptations of transgenic and isogenic plants to the same strictly controlled growth environment. Approximately 100 total proteins resulted differentially modulated in the expression level as a consequence of the environmental influence (WT06 vs WT05), whereas 43 proteins resulted up- or down-regulated in transgenic seeds with respect to their controls (T06 vs WT06), which could be specifically related to the insertion of a single gene into a maize genome by particle bombardment. Transgenic seeds responded differentially to the same environment as compared to their respective isogenic controls, as a result of the genome rearrangement derived from gene insertion. To conclude, an exhaustive differential proteomic analysis allows to determine similarities and differences between traditional food and new products (substantial equivalence), and a case-by-case assessment of the new food should be carried out in order to have a wide knowledge of its features.