Effect of water addition ongluten protein was investigated by using nuclear magnetic resonance (NMR), scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FITR), differential scanning calorimeter (DSC) and rotational rheometer (DHR). The results showed that the amount of water had a significant effect on the hydration, structure and functional properties of gluten protein. With the increase of water addition, the bound water content of gluten protein was decreased significantly from 22.42% to 17.27%, the free water content was increased significantly from 0.04% to 8.25%, and the weakly bound water content was increased first and then decreased, and the water was preferentially converted into bound water and weakly bound water. The gluten protein network had more dense pores and smaller pore size, but when the water was added more than 160%, the strength of the network was weakened. The secondary structure of gluten protein showed that when the water addition amount was not more than 130%, the β-turn angle and the random curl was decreased significantly with the increase of water addition, and the α-helix and β-folding were increased remarkably. When the water addition amount was more than 130%, the secondary structure change of gluten protein was slowed down, and there was no obvious changing rule. The heat denaturation temperature of the protein was firstly increased and then decreased. When the water addition amount reached 150%, the maximum value was 82.4℃, indicating that the thermodynamic stability was increased with the increase of water. The storage modulus G′ and the loss modulus G″of gluten protein all showed a downward trend. After the water addition was more than 130%, the downward trend was slowed down. When the water content was 150%, the elasticity was higher than 140%. The above results indicated that 150% may be the better water content of gluten protein, which also laid a foundation for further revealing the mechanism of water addition to wheat dough. 