AbstractRecent studies have demonstrated that hydrogen sulfide (H2S) produced through the activity of l‐cysteine desulfhydrase (DES1) is an important gaseous signaling molecule in plants that could participate in abscisic acid (ABA)‐induced stomatal closure. However, the coupling of the DES1/H2S signaling pathways to guard cell movement has not been thoroughly elucidated. The results presented here provide genetic evidence for a physiologically relevant signaling pathway that governs guard cell in situ DES1/H2S function in stomatal closure. We discovered that ABA‐activated DES1 produces H2S in guard cells. The impaired guard cell ABA phenotype of the des1 mutant can be fully complemented when DES1/H2S function has been specifically rescued in guard cells and epidermal cells, but not mesophyll cells. This research further characterized DES1/H2S function in the regulation of LONG HYPOCOTYL1 (HY1, a member of the heme oxygenase family) signaling. ABA‐induced DES1 expression and H2S production are hyper‐activated in the hy1 mutant, both of which can be fully abolished by the addition of H2S scavenger. Impaired guard cell ABA phenotype of des1/hy1 can be restored by H2S donors. Taken together, this research indicated that guard cell in situ DES1 function is involved in ABA‐induced stomatal closure, which also acts as a pivotal hub in regulating HY1 signaling.