Haynaldia villosa (2n=14, VV), a wild grass of the subtribe Triticeae, serves as potential gene resources for wheat genetic improvement.  In this study, the proteome characterization during grain development of Yangmai 5 and Yangmai 5-H. villosa 6VS/6AL translocation line was investigated by a comparative proteomic approach.  Two-dimensional electrophoresis identified 81 differentially accumulated proteins (DAPs) during five grain developmental stages in wheat-H. villosa translocation line.  These proteins were mainly involved in stress defense, storage protein, energy metabolism, protein metabolism and folding, carbon metabolism, nitrogen metabolism, and starch metabolism.  In particular, 6VS/6AL translocation led to significant upregulation of 36 DAPs and specific expression of 11 DAPs such as chitinase, thaumatin-like proteins, glutathione transferase, α-amylase inhibitor, heat shock proteins, and betaine aldehyde dehydrogenase.  These proteins mainly involved in biotic and abiotic stress responses.  Further analysis found that the upstream 1 500 promoter regions of these stress-responsive DAP genes contained multiple high-frequency cis-acting elements related to stress defense such as abscisic acid response element ABRE, methyl jasmonate (MeJA)-response element TGACG-motif and CGTCA-motif involved in oxidative stress and antioxidant response element (ARE).  RNA-seq and RT-qPCR analyses revealed the high expression of these stress-defensive DAP genes in the developing grains, particularly at the early-middle grain filling stages.  Our results demonstrated that 6VS chromosome of H. villosa contains abundant stress-defensive proteins that have potential values for wheat genetic improvement.