Grain sorghum (Sorghum bicolor) is a major staple for a large portion of the world. The crop ranks fifth among the cereals world-wide with respect to its importance for food and feed applications. To this end, the grain harvested from sorghum, and the millets provides an important source for dietary calories and protein for approximately one billion people in the semi-arid regions of the world. However, grain sorghum products are known to have relatively poor digestibility, only approximately 50%–70%, in comparison with other grains, such as wheat and maize, which tend to have digestibility percentages over 80% and 70%, respectively. Protein with high digestibility is by definition nutritionally superior owing to the increased availability of amino acids. Digestibility can be impacted by both protein–protein and⁄or protein–nonprotein interactions. However, with respect to grain sorghum, it is thought that the major factor influencing digestibility is the former because of high protein cross-linking around the protein body. To understand the mechanism of seed storage proteins in the sorghum, the proteomic analysis was carried out between the wild(BTX623) and mutant(M271207) genotypes of sorghum. Proteins were separated from the mature seed using IEF in the first-dimension and SDS-PAGE in the second dimension along with hybrid LTQ-FTICR mass spectrometry. After image analysis using Progenesis SameSpot software, we identified the 62 differential expressed protein spots out of 293 protein spots. Out of total differential expressed spots, 35 differential expressed protein spots (more than2-fold) were analyzed by mass spectrometry. Out of 35 protein spots, we were identified 20 protein spots as up-regulated and 15 protein spots as downregulated, significantly. In our proteomic investigation, the candidate proteins may provide novel clues for better understanding the characteristics of seed proteins in Sorghum.