To evaluate the response of alfalfa to water deficit(WD) stress, WD-induced candidates were investigated through a proteomic approach. Alfalfa seedlings were exposed to WD stress for 12 and 15 days respectively, followed by 3 days re-watering. Water deficit increased H2O2 content, lipid peroxidation, DPPH(1,1-diphenyl-2-picrylhydrazyl)-radical scavenging activity, and the free proline level in alfalfa roots. Root proteins were extracted and separated by two-dimentional polyacrylamide gel electrophoresis(2-DE). A total of 49 WD-responsive proteins were identified in alfalfa roots; 25 proteins were reproducibly found to be up-regulated and 24 were down-regulated. Two proteins, namely cytosolic ascorbate peroxidase(APx2) and putative F-box protein were newly detected on 2-DE maps of WD-treated plants. We identified several proteins including agamous-like 65, albumin b-32, inward rectifying potassium channel, and auxin-independent growth promoter. The identified proteins are involved in a variety of cellular functions including calcium signaling, abacisic acid(ABA) biosynthesis, reactive oxygen species(ROS) regulation, transcription/ translation, antioxidant/detoxification/stress defense, energy metabolism, signal transduction, and storage. These results indicate the potential candidates were responsible for adaptive response in alfalfa roots. To evaluate the response of alfalfa to water deficit (WD) stress, WD-induced candidates were investigated through a proteomic approach. Alfalfa seedlings were exposed to WD stress for 12 and 15 days respectively, followed by 3 days re-watering. H2O2 content, lipid peroxidation, DPPH (1,1-diphenyl-2-picrylhydrazyl) -radical scavenging activity, and the free proline level in alfalfa roots. Root proteins were extracted and separated by two-dimentional polyacrylamide gel electrophoresis (2-DE) A total of 49 WD-responsive proteins were identified in alfalfa roots; 25 proteins were reproducibly found to be up-regulated and 24 were down-regulated. Both proteins, including cytosolic ascorbate peroxidase (APx2) and putative F-box protein were newly detected on 2-DE maps of WD-treated plants. We identified several proteins including agamous-like 65, albumin b-32, inward rectifying potassium channel, and auxin-independent growth promoter. The identified proteins are involved in a variety of cellular functions including calcium signaling, abacisic acid (ABA) biosynthesis, reactive oxygen species (ROS) regulation, transcription / translation, antioxidant / detoxification / stress defense, energy metabolism, signal transduction, and storage. responsible for adaptive response in alfalfa roots.