Abstract: TheCa²⁺transduction pathways have been implicated in mediating stress response and tolerance in plants. In order to investigate the mechanism ofCa²⁺signaling mediated by nitric oxide (NO) during seed germination and antioxidation in Medicago sativa under drought stress, sodium nitroprusside (SNP, nitric oxide donor), CaCl2, and methylene blue (NO blockers) and LaCl3 (Ca2+ channel blockers) were used in this study, and alfalfa seeds were pre-soaked with these solutions. An index of germination was markedly decreased under drought stress induced by 15% polyethylene glycol treatment, but this effect was reversed after treatments with SNP and ^Ca2+. Moreover, 0.1 mmol/L SNP or 10 mmol/L CaCl2 alleviated drought stress damage. Compared to SNP or CaCl2 treatment alone, germination rate significantly increased by 8.96% and 19.67% respectively when the seeds were treated with both SNP and ^Ca2+. Furthermore, both SNP and CaCl2 treatments increased content of starch, soluble sugar, soluble protein and proline, and activities of amylase, superoxide dismutase, peroxidase and catalase, whereas malondialdehyde content decreased compared to that under SNP or CaCl2 treatment alone. The changes were slower for seeds receiving both NO andCa²⁺treatments than for NO orCa²⁺treatment alone. Interestingly, with added exogenousCa²⁺and methylene blue, the promotional effect ofCa²⁺was inhibited. In addition, the promotional effect of NO was inhibited by La3+. This indicates that NO mediated protein modifications in alfalfa seeds under drought stress through theCa²⁺signaling pathway.