SummarySeed storage proteins are synthesized as sources of carbon, nitrogen and sulfur for the next generation of plants. Their composition changes according to nutritional conditions. Here, we report the precise molecular identification of seed proteins by proteomic analysis of wild‐type Arabidopsis thaliana and methionine‐over‐accumulating mutant mto1‐1 plants. The identities of 50 protein spots were determined in the protein extract of mature Arabidopsis seeds by two‐dimensional (2D) gel electrophoresis and subsequent mass spectrometric analysis. Of these protein spots, 42 were identified as derived from 12S globulins or 2S albumins. These results indicate that approximately 84% of protein species in Arabidopsis seeds are derived from a few genes coding for 12S globulins and 2S albumins. Extensive mass spectrometric analysis of the 42 spots revealed that successive C‐terminal degradation occurred on the 12S globulins. The feasibility of this C‐terminal processing was rationalized by molecular modeling of the three‐dimensional structure of 12S globulins. The C‐terminal degradation at glutamic acid residues of the 12S globulin subunits was repressed under sulfur‐deficient conditions. Transcriptome analysis was combined with proteomic analysis to elucidate the mechanism of changes in seed protein composition in response to sulfur deficiency. The results suggest that seed storage proteins in Arabidopsis undergo multi‐layer regulation, with emphasis on post‐translational modifications that enable the plant to respond to sulfur deficiency.