Abstract: To explore the role of H₂S signal in regulating stomatal movement in alfalfa and the relationship between H₂S and Ca²⁺ during this process, wild type and the calcium transporter mutants of Medicago truncatula were used as experimental materials, and the transcriptional level, cellular level and physiological level were studied respectively, and the expression levels of related genes were compared by qRT-PCR, the content of Ca²⁺in vivo was detected by fluorescent probe, the content of H₂S was measured by electrode method, and the stomatal aperture was observed by microscope respectively. The results showed that compared with the wild type, the content of H₂S in the mutant NF3011 and NF2734 decreased significantly; the H₂S signal inhibited the expression of calcium transporter encoding gene MTR＿6g027580, the exogenous physiological concentration of H₂S fumigation could induce stomatal closure of Medicago truncatula, and the combination of treatment with LaCl3, Ca²⁺ channel blocker, had no effect on stomatal movement of wild type, but opposite in mutant; the concentration of Ca²⁺ in guard cells was measured by fluorescence probe, which were consistent with the change law of stomatal aperture. In conclusion, the content of Ca²⁺ in guard cells of leaves is induced by H₂S, whose stomatal aperture become smaller at the same time. In this process, the change of intracellular Ca²⁺concentration mainly depend on Ca²⁺ transporter, and small partly depend on Ca²⁺ channel. The results of this study not only enrich the mechanism of H₂S signaling in theory, but also have the potential to be applied to alfalfa production and other crops.