A stoma forms by a series of asymmetric divisions of a stomatal lineage precursor cell and the terminal division of a guard mother cell (GMC). The symmetric division of the GMC is rigidly restricted to only once through complex genetic regulation mechanisms. Here, we show that nitric oxide (NO) is involved in the regulation of the GMC terminal division. NO donor treatment results in the formation of single guard cells (SGCs). SGCs are also produced in plants that accumulate high NO, whereas clustered guard cells (GCs) appear in plants with low NO accumulation. NO treatment promotes the formation of SGCs in the stomatal cell signaling mutants sdd1, epf1 epf2, tmm1, erl2 and yda-1, reduces the cell number per stomatal cluster in the fama-1 and flp-1 myb88, but has no effect on stomatal cells of cdkb1;1 cyca2;2 cyca2;3 cyca2;4 quadruple mutants. Aminocyclopropane-1-carboxylic acid (ACC), a positive regulator of GMC division, reduces the NO-induced SGC formation. Further investigation found that NO inhibits ACC synthesis by repressing the expression of several ACC SYNTHASE ( ACS) genes, and in turn ACC represses NO accumulation by promoting the expression of HEMOGLOBIN 1 ( HB1) which encodes a NO scavenger. This work shows that NO plays a role in the regulation of the GMC terminal division by modulating ACC accumulation in the Arabidopsis cotyledon.