Through pharmacological combined with laser scanning confocal microscope (LSCM) and spectrophotography to study the role of Ca2+ and NO in signaling during Vicia faba L. stomatal movement response to ethylene (Eth). The results showed that treatment with ethephon (0.004%, 0.04%, 0.4%) resulted in a time- and dose-dependent stomatal closure under light. NO scavenger cPTIO, nitrate reductase inhibitor NaN3, or extracellular Ca2+ chelation EGTA reduced ethylene-induced stomatal closure. Moreover, ethylene was shown to enhance nitric oxide levels and, corresponding, nitrate reductase activity. Inhibition of the nitrate reductase diminished ethylene-induced NO production in both stomatal guard cell and leaf. Finally, ethylene-induced NO levels and nitrate reductase activity decreased when Ca2+ was compromised. On the basis of biochemical and pharmacological experimental results, we can conclude that Ca2+ and NO were involved in the signal transduction pathway of ethylene induced stomatal closure. Nitrate reductase-derived NO may represents a novel downstream component of Ca2+ signaling cascade during ethylene-induced stomatal movement in Vicia faba L.