Phytohormone salicylic acid (SA) plays important roles in plant responses to environmental stress. However, knowledge about the molecular mechanisms for SA affecting the stomatal movements is limited. In this paper, we demonstrated that exogenous SA significantly induced stomatal closure and nitric oxide (NO) generation in Arabidopsis guard cells based on genetic and physiological data. These effects were significantly inhibited by the NO scavenger c-PTIO, NO synthase (NOS) inhibitor L-NAME or nitrate reductase suppressor tungstate respectively, implying that NOS and nitrate reductase (NR) participate in SA-evoked stomatal closing. Furthermore, the effects of SA promotion of stomatal closure and NO synthesis are significantly suppressed in NR single mutants of nia1, nia2 or double mutant nia1/nia2, compared with the wild type plants. This suggests that both Nia1 and Nia2 are involved in SA-stimulated stomatal closure. In addition, pharmacological experiments showed that protein kinases, cGMP and cADPR are involved in SA-mediated NO accumulation and stomatal closure induced by SA in Arabidopsis.