Hydrogen sulfide (H2S) is a gas produced endogenously in organisms from the three domains of life. In mammals, it is involved diverse of physiological processes including regulation of blood pressure, and plays an important role in memory. In contrast, in unicellular organisms the physiological role of H2S has not been studied in detail. In yeast, for example, in the winemaking industry H2S is an undesirable byproduct because of its rotten egg smell, however its biological relevance has never been evaluated. The effect of H2S in cells is linked to a post-translational modification in cysteine residues known as S-persulfidation. We evaluated S-persulfidation in the Saccharomyces cerevisiae proteome. We screened S-persulfidated proteins from cells growing in fermentable carbon sources and we identified several glycolytic enzymes as S-persulfidation targets. Pyruvate kinase, catalyzing the last irreversible step of glycolysis, increases its activity in presence of a H2S donor. Yeast cells treated with H2S increased ethanol production; also, mutant cells that endogenously accumulate H2S produced more ethanol and ATP during the exponential growth phase. This mechanism of regulation of metabolism seems to be evolutionarily conserved in other yeast species because H2S induces ethanol production in the pre-Whole Genome Duplication Kluyveromyces marxianus and Meyerozyma guilliermondii. Our results suggest a new role of H2S in regulation of metabolism that could be useful for biotechnological applications.