Herein, the physical migration and chemical conversion of phytosterols during the alkali neutralisation process of corn oil were investigated with molecular insights. A significant decrease in the phytosterol content was attributed to the migration from oil to the soapstock, along with slight chemical conversions between free phytosterols and phytosteryl esters in the heterogeneous system. Moreover, the migration of phytosterols was positively correlated with the water content in a model system through low-field nuclear magnetic resonance spectroscopy, which was attributed to the hydrogen bonding between free phytosterols and water. Compared to sodium oleate and sodium linoleate, sodium palmitate caused a higher loss of phytosterols due to its low solubility. X-ray diffraction analysis confirmed the formation of a bilayer structure of the sodium salt of fatty acids, suggesting the aggregation of the sodium salt of fatty acids and phytosterols at neutralisation temperatures. Small-angle X-ray scattering analysis suggested that the concentration of the NaOH solution affected the structure of sodium oleate and the area of the polar head base, which was associated with the loss of free phytosterols. The study presents an important fundamental scientific basis for the retention of phytosterols toward the development of more healthy and nutritious edible oils.