Ca2+, an ubiquitous second messenger in the signal transudation pathway, is required for various physiological and developmental processes in plant. Jasmonic acid (JA) has been known to induce the stomatal closure. By monitoring the changes of [Ca2+]cyt with fluorescent probe Fluo-3 AM under the confocal microscopy, we observed that exogenous JA increased [Ca2+]cyt in guard cells of Vicia faba L. while the control and linolenic acid (LA), which is a precursor of JA, could hardly affect the change of [Ca2+]cyt. EGTA, a chelator of Ca2+ completely blocked JA-induced stomatal closure. After epidermis pretreated with EGTA, JA failed to result in [Ca2+]cyt increasing. Ruthenium red that blocked Ca2+ released from intracellular Ca2+ store could not significantly change JA-induced stomatal closure, while JA still increased [Ca2+]cyt. Furthermore, Ca2+ channel inhibitor of nifedipine (NIF) reduced the effectiveness of JA-induced stomatal closure and JA-induced increasing fluorescent intensity in guard cells. The results demonstrated that Ca2+ is involved in the signal transduction of JA induced stomatal closure, and the source of [Ca2+]cyt increasing in guard cells induced by JA might derive mainly from the external stores.