The roles of copper amine oxidase (CuAO) and its products hydrogen peroxide (H2O2) during ethylene-induced stomatal closure in Vicia faba were investigated. 2-chloroethylene phosphonic acid (ethephon), an ethylene-releasing compound, stimulated apoplast CuAO activity, increased H2O2 production in guard cells and induced stomatal closure. These effects were suppressed by aminoguanidine(AG) and 2-bromoethylamine(BEA), two irreversible inhibitors of CuAO. In the metabolized products of CuAO, only H2O2 could prevent the inhibitory effect of AG or BEA on stomatal closure induced by ethephon. Putrescine (Put), a main substrate of CuAO, did not prevent ethylene-induced stomatal closure. Take together, the present data suggest that H2O2 generated by CuAO involves in ethylene-induced stomatal closure in Vicia faba.