Hydrogen sulfide (H₂S), produced by cystathionine γ-lyase (CSE), acts as a physiologic vasorelaxant. This study shows that H₂S covalently modifies cysteine residues in proteins through S-sulfhydration, a posttranslational modification that enhances the activity of proteins such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and promotes actin polymerization. Sulfhydration is a physiologic process that occurs in the liver, where about 10-25% of proteins, including GAPDH, β-tubulin, and actin, are sulfhydrated under physiological conditions. Sulfhydration is reversible by reducing agents like dithiothreitol (DTT) and is distinct from S-nitrosylation, which typically reduces cysteine reactivity. The study also demonstrates that CSE is a major source of H₂S in the liver, and its absence in CSE⁻/⁻ mice leads to reduced H₂S production and impaired sulfhydration of proteins. Sulfhydration of GAPDH occurs primarily at cysteine 150, which is critical for its catalytic activity. Sulfhydration of GAPDH increases its catalytic activity, while sulfhydration of actin enhances its polymerization. These findings suggest that S-sulfhydration is a physiologic signaling mechanism that influences various biological processes, including vascular relaxation, inflammation, and cell signaling. The study also highlights the potential of S-sulfhydration as a regulatory mechanism for protein function in multiple biological pathways.Hydrogen sulfide (H₂S), produced by cystathionine γ-lyase (CSE), acts as a physiologic vasorelaxant. This study shows that H₂S covalently modifies cysteine residues in proteins through S-sulfhydration, a posttranslational modification that enhances the activity of proteins such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and promotes actin polymerization. Sulfhydration is a physiologic process that occurs in the liver, where about 10-25% of proteins, including GAPDH, β-tubulin, and actin, are sulfhydrated under physiological conditions. Sulfhydration is reversible by reducing agents like dithiothreitol (DTT) and is distinct from S-nitrosylation, which typically reduces cysteine reactivity. The study also demonstrates that CSE is a major source of H₂S in the liver, and its absence in CSE⁻/⁻ mice leads to reduced H₂S production and impaired sulfhydration of proteins. Sulfhydration of GAPDH occurs primarily at cysteine 150, which is critical for its catalytic activity. Sulfhydration of GAPDH increases its catalytic activity, while sulfhydration of actin enhances its polymerization. These findings suggest that S-sulfhydration is a physiologic signaling mechanism that influences various biological processes, including vascular relaxation, inflammation, and cell signaling. The study also highlights the potential of S-sulfhydration as a regulatory mechanism for protein function in multiple biological pathways.