The current study investigated the effects of various freeze-thaw (F-T) cycles on gel properties and molecular forces involved in gelation of soy protein isolate (SPI). The gel strength of SPI submitted to 4 and 5 freeze-thaw (F-T) cycles increased 1.95- and 1.98 folds compare to control, respectively (P<0.05). The observation from scanning electron microscope and atomic force microscopy indicated that the SPI submitted to 4 and 5 F-T cycles exhibited smoother, flatter and denser gel surface morphology images than the control samples. G' and G '' values of the sample submitted to 4 and 5 F-T cycles dramatically increased during the cooling period in dynamic rheological testing and the final G' value increased 32.6-folds for the samples submitted to 5 F-T cycles compare to control. In addition, the results of particle size distribution provided direct evidence that the aggregation occurred in SPI was accelerated by multiple F-T cycles. The results of molecular force measurement implied that these effects could be primarily attributed to the stronger molecular interactions in SPI gel, with the main forces being the disulfide bond, the hydrogen bond and the hydrophobic interactions. Overall, multiple F-T treatments offered a new way to improve the gel properties of SPI. (C) 2016 Elsevier Ltd. All rights reserved.