Soil salinity is a critical environmental problem that disrupts plant metabolic homeostasis and requires adjustment of metabolic pathways in plant cells, a process that is usually referred to as acclimation. Metabolomics technologies have made significant contributions to the study of plant stress biology through identification of different compounds, such as signaling molecules and metabolic by-products in response to stress conditions, as well as small molecules involved in the plant acclimation processes. Thellungiella salsuginea is a close relative of Arabidopsis thaliana. It has been proposed as an ideal model for studying molecular mechanisms of salinity tolerance in plants because of its 'extremophile’ characteristics manifested by extreme tolerance to high salt conditions. To obtain a better understanding of the molecular mechanisms underlying the response of T. salsuginea to salt stress, an Nuclear Magnetic Resonance (NMR)-based metabolic profiling approach was used to profile metabolite changes in T. salsuginea after treatment with 150 mmol/L or 300 mmol/L NaCl for 24 h. In general, metabolomic studies should be designed to detect as many metabolites as possible in an organism, and a solvent that can extract a diverse group of metabolites should be employed. In our experiments, metabolites were extracted from the leaf tissues using a solvent system of methanol/water (1/1), known to extract many different metabolites. ¹H-NMR spectroscopy of samples from control, 150 mmol/L and 300 mmol/L NaCl treatment was performed separately. Spectral data were analyzed and interpreted using multivariate statistical analyses. Overall, the original NMR spectra were dominated by one of the organic acids, malate (δ 2.38 and 2.68), which is a T. salsuginea metabolite to maintain the osmotic balance in the cells and was approx. 10-100 times higher than other metabolites found in the ¹H-NMR spectra. Principal components analysis (PCA) was used in this work for the separation of control from different concentrations of NaCl-treated groups. PCA is an exploratory unsupervised pattern recognition method since it calculates inherent variation within the data sets without use of the class membership. Following PCA, the PC scores plot indicated highly significant separations (P<0.05) between control and salt treatments, suggesting huge differences in metabolite profiles between control and NaCl-treated samples. In order to understand the origin of the differences between the control and NaCl treatments, the metabolites were identified and quantified. A total of 23 intracellular metabolites, comprising 11 amino acids, 4 sugars, 6 organic acids and 2 others were identified in leaf extracts of T. salsuginea. All of the 23 metabolites showed reproducible and statistically significant changes in NaCl-treated samples compared to control samples. Most metabolites exhibited significant increases in response to NaCl treatment except aspartate and fumarate. To evaluate the connections of these metabolic changes under salt stress, the measured metabolite variations were mapped onto the plant biosynthetic pathways. Metabolites involved in carbohydate metabolism, amino acid biosynthesis, TCA cycle and betaine biosynthesis played an important role in response to salt stress. It is notable that a potentially powerful strategy of salt tolerance in the halophyte T. salsuginea is to increase the TCA cycle intermediates and accumulate the osmoregulatory solute proline. This information, together with quantitative kinetic indices, can be used to model and simulate metabolic pathways. This study has further highlighted the value of the metabolic profiling approach in discovering and validating plant metabolic mechanisms in response and acclimation to salt stress.