Soil salinization is one of the most common abiotic stresses affecting plant growth and is becoming an important issue, owing to its impact on agricultural production and the environment. Saline-alkali soil is becoming particularly widespread and may cover more than 50% of all arable lands by the year 2050. The combination of soil salinization and high pH conditions represents a major impediment for plant growth and crop productivity. The study area was a typical alkalinized grassland in northeastern China. Jerusalem artichoke (Helianthus tuberosus) is an annual flowering plant that has been cultivated as a vegetable, fodder crop, and bioenergy material in many countries, owing to its high levels of polysaccharides, especially inulin. The ability to survive in the saline-alkali soils of semiarid areas is one of the most important characters of Jerusalem artichoke. Jerusalem artichoke tubers were sown in farmland, light saline-alkali, or severe saline-alkali soil and sprouting tubers were collected from the sample in May of the following year. The malondialdehyde, free proline, and soluble sugar contents, antioxidant enzyme activity, and protein profile were quantified, in order to assess the physiological response of Jerusalem artichoke to saline-alkali stress. The soil electrical conductivity (0-20 cm) indicated that the soluble salt contents of the three soil types were significantly different, with the lowest soluble salt content in the farmland soil and the greatest content in the severe saline-alkali soil. With increasing soluble salt content, the malondialdehyde content increased, obviously indicating higher levels of stress, and increased free proline content indicated that Jerusalem artichoke could enhance its resistance to salt stress via osmotic adjustment. To investigate the proteomic response of the sprouting tubers to saline-alkali stress, two-dimensional gel electrophoresis (2-DE) gels from three biological experiments were analyzed. Then, using ImageMaster 2D Platinum Software, we identified more than 1000 highly reproducible protein spots on the coomassie brilliant blue (CBB)-stained 2D gels. A total of 80 differentially expressed proteins were successfully identified using MALDI-TOF/TOF analysis, according to the peptide matching results provided by MASCOT. Among these proteins, 42 were detected in the sprouting H. tuberosus tubers from the light saline-alkali soil and 38 of the proteins accumulated differentially in the tubers grown in the severe saline-alkali soil. KEGG pathway analysis attributed these proteins to eleven different metabolic pathways, which included carbohydrate and polysaccharide metabolism (15%), energy metabolism (7.5%), genetic information processing (28.75%), amino acid metabolism (11.25%), nucleotide metabolism (2.5%), biosynthesis of secondary metabolites (3.75%), signal transduction (17.5%), transport and catabolism (2.5%), cell motility (2.5%), and unknown (8.75%). The differentially expressed proteins were mainly involved in genetic information processing, which might indicate that the regulation of proteins involved in DNA replication, transcription, protein synthesis, and protein folding are responsive to saline-alkali stress and play a pivotal role in salinity tolerance. Carbohydrate, energy, and amino acid metabolism-related proteins constituted one-third of the differentially expressed proteins, which suggests that metabolism homeostasis is important for the survival of seedlings exposed to saline-alkali stress. These findings provide new insight into the underlying molecular mechanisms of saline-alkali resistance in Jerusalem artichoke.