The role of ethylene and the relationship between ethylene and nitric oxide (NO) in ultraviolet B (UV-B)-induced stomatal closure were investigated in Vicia faba L. (broad bean) plants by epidermal strip bioassay, laser-scanning confocal microscopy and assay of ethylene production. In response to UV-B radiation, the rise of NO level in guard cells was after ethylene evolution peak, but preceded stomatal closure. Both UV-B-induced NO generation in guard cells and subsequent stomatal closure were substantially inhibited not only by NO scavenger and nitrate reductase (NR) inhibitors, but also by interfering with ethylene synthesis or perception. Although exogenous NO could reverse the inhibitive effect of interfering with ethylene synthesis or perception on UV-B-induced stomatal closure, the inhibitive effect of NO scavenger and NR inhibitors on UV-B-induced stomatal closure could not be rescued by exogenous ethylene. Taken together, our results clearly show that ethylene participates in the UV-B-induced stomatal closure and acts upstream of the NR source of NO generation in V. faba.