NAD kinase2 (NADK2) plays key roles in chloroplastic NADP biosynthesis, stress adaptation and modulation of cellular metabolisms in Arabidopsis. However, it is unknown whether and how NADK2 affects abscisic acid (ABA)-mediated stomatal movement. Here, we detected that null mutant nadk2 was more sensitive to drought stress than WT, and NADK2 gene was active in guard cells. Furthermore, NADK2 mutation impaired ABA-induced stomatal closure and ABA inhibition of light-promoted stomatal opening. NADK2 disruption also impaired ABA-stimulated accumulation of HO, Ca and nitric oxide (NO) in guard cells, but did not affect the stomatal closure evoked by exogenous HO, Ca or NO. Expression analysis revealed that ABA-promoted increases in transcripts of AtrbohD, AtrbohF and NIA1 were markedly arrested in guard cells of nadk2 compared with those of WT. Besides, genetic evidence indicated that NADK2 acted synergistically with OST1 and ABI1 during ABA-induced stomatal closure. Together, these results suggest that NADK2 is an essential positive regulator, and functions upstream of HO in guard cell ABA signaling. It stimulates stomatal closure mainly through increasing the generation of HO, Ca and NO in guard cells in Arabidopsis.