Hypoxia caused by waterlogging results in a severe loss of crop production. At the primary stage of wheat development, the seminal roots have strategies to survive under hypoxia through alternative metabolism coupling root anatomical modification. The present study used a model system of lysigenous aerenchymatous seminal roots from a representative seedling stage of wheat to elucidate the root physiology in response to soil hypoxia. Seminal roots characteristic with lysigenous aerenchyma tissues were developed in pot cultures for 7 days under two hypoxic conditions, water depths of 15 cm below and 3 cm above the soil surface. Proteins from the roots were separated using two-dimensional polyacrylamide gel electrophoresis and identified using mass spectrometry. The results showed that approximately 345 distinct protein spots were detected by 2-DE, 29 spots changed in the expression levels between the control and two hypoxic plants, and 10 spots exhibited a reproducible up- or down regulated fluctuation. The up-regulated proteins were thought to be involved in alteration in energy and redox status, defense responses and cell wall turnover. These results suggest the effects of soil hypoxia on the activity of the identified up-regulated proteins and their roles in alternative respiration and cell degeneration in wheat in order to gain metabolic adjustment under hypoxia stress.