BACKGROUND: Early seedling development is a critical step in the establishment of subclover (Trifolium subterraneum), an economically important and widespread pasture legume. In this study the seed storage proteome of this non-model species was characterised in mature dry seeds and during imbibition by using two-dimensional electrophoresis coupled with tandem mass spectrometry. RESULTS: The phenol-extracted proteome of subclover dry seeds consisted of 97 polypeptide spots displayed within a window of pI 3-10 and molecular mass 10-150 kDa. De novo sequencing coupled with MS BLAST search enabled the confident identification of 61 proteins, which corresponded to 59 7S vicilin- and two 11S legumin-type globulins. The experimental mobility of vicilin isoforms along with peptide mapping indicated that low-molecular-mass polypeptides might account for the post-translational proteolysis of small vicilin subunits according to the model described for those of pea. Analysis of quantitative changes in the seed storage proteome upon imbibition showed that vicilin catabolism according to a site-specific process was favoured during early seedling growth in T. subterraneum. CONCLUSION: The establishment of a seed proteome map for T. subterraneum pointed to vicilins as dominant proteins in mature seeds whose catabolism features during early seedling growth may be of relevance under environmental conditions.