Abstract Glycerol plasticized soy protein is an important kind of biodegradable materials among the possible soy protein systems. Fully realized performance relies on the investigation and understanding of the molecular structure of the plasticized soy protein. However, the relevant research is primarily limited in the microstructure and physical properties of the mixtures with a lack of knowledge on the intermolecular interactions at molecular and nano-scale levels. In this work, intermolecular interaction and molecular motion of soy protein in glycerol plasticized systems were investigated at molecular and nanometer levels. Several techniques including atomic force microscope (AFM), transmission electron microscope (TEM) and 13C solid-state nuclear magnetic resonance (13C NMR), have been effectively employed. Observations from AFM and TEM indicated that the glycerol plasticized soy protein materials were heterogeneous at nanometer scale and there were two important phases existing in the materials, i.e. protein-rich domain and glycerol-rich domain. The protein-rich domain was composed of compact aggregations of pan-like protein while the glycerol-rich domain possessed a loose chain-like structure. Further studies on the interactions between soy protein and glycerol at molecular level using NMR indicated that the strong hydrogen bonds between glycerol and protein molecules was greatly affected by amino acid residues on protein chains. The protein-rich micro-domains were mainly composed of amino acids with long alkane lateral chains or aromatic ring lateral groups, while amino acids with polar groups or short nonpolar lateral groups formed glycerol-rich micro-domains. These results would be beneficial for the development and realization of plasticized soy protein based nanocomposites and blend materials with high performance.