Abstract: Glycerol is a simple polyol compound, it has many biological and industrial applications when mixed with water. It can stabilize proteins under extreme conditions, such as heat stress, cold shock, high pressure, etc. Most proteins are usually stored in a buffer system with the presence of glycerol after purification to maintain the stability of their biological activity. At present, there are still some controversies about the protective mechanism of glycerol on proteins. Most studies were focused on the effect of high concentrations of glycerol on protein structure. However, there were few related studies on low concentrations of glycerol. Therefore, an in-depth understanding of the effects of low concentrations of glycerol on protein structure is beneficial to protein-related research and applications. Electrospray ionization-mass spectrometry (ESI-MS) has become a commonly used technique to study protein structure and function, which is reliable and time-efficient. Protein conformational change can be described in more detailed by ESI combined with ion mobility spectrometry. Ion mobility provides an additional dimension of resolution. When ions with different sizes, charges and shapes pass through the chamber, they are migrated and separated in neutral media according to the collision cross-section (CCS). By measuring the molecular weight, charge state distribution, drift time and CCS, the structural change information of the protein can be obtained. In this study, bovine serum albumin (BSA) was used as a model protein, and ion mobility-mass spectrometry (IM-MS) and dynamic light scattering were used to investigate the effect of low concentration glycerol on protein structure under approximate physiological conditions. The results showed that glycerol within a certain concentration range could cause an increase in the average charge and CCS of BSA. Meanwhile, a single charge state of BSA also experienced an increase of CCS in presence of glycerol. In addition, the hydration radius of BSA were increased with the increase of glycerol concentration. The results indicated that low concentrations of glycerol can loosen proteins. It is interesting to note that the unfolding voltage of BSA containing 0.5% glycerol was higher than BSA without glycerol and the unfolding transition occurred in a wider voltage range, indicating that low concentration of glycerol can improve the stability of the protein. These results provide further insight into the mechanism of glycerol stabilizing proteins and may also provide references for the related research and development of proteins.