Abstract: This study investigates the protective effect of wheat antifreeze proteins (WAFPs) on the freeze-thaw stability of gluten proteins. WAFPs were added to dough with samples without WAFPs serving as the control. Both sample sets underwent 2, 4, 6, 8, and 10 freeze-thaw cycles, each comprising 2 h of freezing at -40 ℃, 20 h of storage at -20 ℃, and 2 h of thawing at 25 ℃. Following the freeze-thaw treatments, gluten proteins were extracted by washing with distilled water. The measured parameters included water-holding capacity, oil-holding capacity, emulsifying capacity, emulsification stability, secondary structure (analyzed by Fourier-transform infrared spectroscopy), water distribution (assessed by low-field nuclear magnetic resonance), free thiol content, microstructure (observed via scanning electron microscopy), rheological properties (elastic modulus G', viscous modulus G″, and loss tangent tanδ), and SDS-PAGE subunit composition. The results indicate that WAFPs significantly mitigate freeze-thaw-induced degradation of gluten proteins. Compared with the control, WAFPs reduced the exposure of hydrophobic groups, inhibited the decline in water-holding capacity and the increase in oil-holding capacity, and enhanced emulsifying properties and stability. Furthermore, WAFPs bind to gluten proteins via hydrogen bonding and hydrophobic interactions, leading to a smaller reduction in random coils and α-helices, less variation in free and bound water content, a significant decrease in free thiol content, a more regular network structure, increased elastic and viscous moduli, and reduced dissociation of certain high-molecular-weight protein subunits. In summary, WAFPs exert multifaceted protective effects on wheat gluten proteins during freeze-thaw cycling, demonstrating potential for improving the quality of frozen dough products.