Abstract: To explore methanol-induced conformational change of lysozyme at pH 2.0, electrospray ionization mass spectrometry (ESI-MS) and circular dichroism (CD) were employed to probe the conformations of lysozyme solutions containing different concentrations of methanol. The charge state distribution (CSD) of lysozyme didn’t change significantly before the methanol concentration rose up to 70%. However, when it reached 75%, the high CSD centered at 16+ began to emerge, and bimodal was observed, suggesting that the tertiary structure of lysozyme was destroyed. Meanwhile, CD spectra showed that the percentage of helical state increased obviously, suggesting the secondary structure was partially affected. The results indicated when methanol concentration was over 75%, the conformation of lysozyme could change from folded into unfolded, accompanied by the collapse of tertiary structure. Differential scanning calorimetry (DSC) indicated temperature peak value (T_m) rose up from 79.6 ℃ to 81.8 ℃ after the solid lysozyme dissolved in 5% methanol solution, which suggested thermal stability of lysozyme was intensified.