Recent 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 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.