Proteomics has become a powerful technique for investigating unintended effects in genetically modified crops. In this study, we performed a comparative proteomics of the seeds of phytase-transgenic (PT) and non-transgenic (NT) maize using 2-DE and iTRAQ techniques. A total of 148 differentially expressed proteins (DEPs), including 106 down-regulated and 42 up-regulated proteins in PT, were identified. Of these proteins, 32 were identified through 2-DE and 116 were generated by iTRAQ. It is noteworthy that only three proteins could be detected via both iTRAQ and 2-DE, and most of the identified DEPs were not newly produced proteins but proteins with altered abundance. These results indicated that many DEPs could be detected in the proteome of PT maize seeds and the corresponding wild type after overexpression of the target gene, but the changes in these proteins were not substantial. Functional classification revealed many DEPs involved in posttranscriptional modifications and some ribosomal proteins and heat-shock proteins that may generate adaptive effects in response to the insertion of exogenous genes. Protein-protein interaction analysis demonstrated that the detected interacting proteins were mainly ribosomal proteins and heat-shock proteins. Our data provided new information on such unintended effects through a proteomic analysis of maize seeds.