This study investigated the impact of multi freeze-thaw cycles on emulsifying activity and structural characteristics of soy protein isolate (SPI). The emulsifying activity index and emulsion stability index of the SPI submitted to 3,4 and 5 freeze-thaw cycles were significantly higher than that of native protein (P < 0.05). Light microscopy provided consistent data supporting the emulsion results. Total sulfhydryl groups reduced from 15.2 mu mollg in the control to 11.2 mu mol/g in the SPI following 3 freeze-thaw cycles. Freeze-thaw cycles significantly increased surface hydrophobicity (P < 0.05) as the number of freeze-thaw cycles increased from 1 to 3. In addition, the results of circular dichroism spectra, UV-vis spectra, intrinsic fluorescence spectroscopy and high-performance size exclusion chromatography provided indirect evidence that some of high molecular weight molecules were broken into lower molecular weight molecules as a result of water redistribution, ice recrystallization and the protein mechanical damage as water turned into ice during the freeze-thaw cycles. (C) 2015 Elsevier Ltd. All rights reserved.