Development of new technologies for evaluating genetically modified (GM) crops has revealed that there are unintended insertions and expression changes in GM crops. Profiling techniques are non-targeted approaches and are capable of detecting more unintended changes in GM crops. Here, we report the application of a comparative proteomic approach to investigate the protein profile differences between a GM rice line, which has a lysine-rich protein gene, and its non-transgenic parental line. Proteome analysis by two-dimensional gel electrophoresis (2-DE) and mass spectrum analysis of the seeds identified 22 differentially expressed protein spots. Apart from a number of glutelins that were detected as targeted proteins in the GM line, the majority of the other changed proteins were involved in carbohydrate metabolism, protein synthesis and stress responses. These results indicated that the altered proteins were not associated with plant allergens or toxicity.