Hydrogen sulfide (H₂S), a physiological vasorelaxant, acts through S-sulfhydration, a posttranslational modification that converts cysteine -SH groups to -SSH. This study demonstrates that H₂S physiologically sulfhydrates a large number of proteins, including actin, tubulin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), in the liver. Sulfhydration enhances GAPDH activity and actin polymerization. In *CSE*^−/−^ mice, which lack endogenous H₂S production, sulfhydration of these proteins is absent. Sulfhydration of GAPDH occurs at Cys150, a residue critical for its catalytic activity. H₂S increases GAPDH activity in a dose-dependent manner, and this effect is reversed by the reducing agent DTT. Sulfhydration also enhances actin polymerization in vitro and in HEK293 cells, an effect that is reversible with DTT. These findings suggest that sulfhydration is a physiologic signal that influences various biological pathways.Hydrogen sulfide (H₂S), a physiological vasorelaxant, acts through S-sulfhydration, a posttranslational modification that converts cysteine -SH groups to -SSH. This study demonstrates that H₂S physiologically sulfhydrates a large number of proteins, including actin, tubulin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), in the liver. Sulfhydration enhances GAPDH activity and actin polymerization. In *CSE*^−/−^ mice, which lack endogenous H₂S production, sulfhydration of these proteins is absent. Sulfhydration of GAPDH occurs at Cys150, a residue critical for its catalytic activity. H₂S increases GAPDH activity in a dose-dependent manner, and this effect is reversed by the reducing agent DTT. Sulfhydration also enhances actin polymerization in vitro and in HEK293 cells, an effect that is reversible with DTT. These findings suggest that sulfhydration is a physiologic signal that influences various biological pathways.