Hydrogen sulfide (H2S) is well known as a potent vasodilator and signaling molecule under normal and pathophysiological conditions. Previously, we reported that in hyperglycemia (HG), impairment of cystathionine β synthase/ cystathionine lyase (CBS/CSE) decreases H2S production leading to vascular remodeling. Treatment with H2S mitigated the adverse effects; however, its precise mechanism was unknown. Literature reports suggest that hyperglycemia suppresses adenosine monophosphate-activated protein kinase (AMP-K) pathway and increases autophagy leading to extracellular matrix (ECM) remodeling. Liver kinase B1 (LKB1) is a protein kinase which has been reported to control the activity of AMP-K. Activation of LKB1, in turn, is regulated by two pseudokinases, STE-20 related protein (STRAD) and mouse protein-25 (MO25). In this study, we hypothesized that H2S mediates its effects by the formation of a tri-molecular complex (STRAD/MO25/LKB1) leading to the activation of AMP-K and modulation of autophagy. Mouse glomerular endothelial cells were used in this study. Under normoglycemic (NG) and HG conditions, cells were treated without or with H2S and activators/inhibitors of the signaling molecules above. The expression of CBS/CSE was diminished by 3-fold under HG compared to NG. H2S production was also decreased [3.5 vs. 7.2 μM/L (HG vs. NG)]. HG decreased LKB1 by 5-fold and AMP-K by 14-fold compared to NG. Autophagy marker LC3A/B ratio was similarly decreased in HG. The expression of periostin, a marker of renal disease progression, was higher (Relative intensity (RI) = 1.9 ± 0.64) in HG compared to NG (RI= 1.4 ± 0.18). In HG, administration of H2S activated STRAD by 4-fold and MO25 by 3-fold causing concomitant phosphorylation of LKB1 (5-fold increase) eventually leading to AMP-K phosphorylation (13-fold increase). Interestingly, phosphorylated AMP-K inhibited autophagy and reduced ECM protein accumulation. Our findings suggest that the protective effect of exogenous H2S in hyperglycemia is mediated by AMP-K activation involving STRAD and MO25 pseudokinases.