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Hydrogen sulfide (HS) is a respiratory poison and also a product of our own metabolism. The toxicity of HS is mitigated by the activity of mitochondrial sulfide quinone oxidoreductase (SQOR), which oxidizes HS while concomitantly reducing coenzyme Q. An unusual cysteine trisulfide cofactor distinguishes SQOR from other members of the flavin disulfide reductase superfamily. In the opening step of the catalytic cycle, an estimated 10-fold rate enhancement is afforded by nucleophilic addition of the sulfide anion to the trisulfide versus a disulfide cofactor. The source of the bridging sulfane sulfur in the trisulfide and its mechanism of installation are, however, unknown. We report that HS exposure (100 ppm corresponding to 20 μM dissolved HS, 24 h) increases SQOR activity five- and twofold in human colon adenocarcinoma (HT-29) and transformed endothelial (EA.hy926) cells, respectively. Since activation is not accompanied by a corresponding increase in SQOR protein levels, we conclude that it involves a post-translational mechanism. CRISPR knockdowns of the sulfurtransferases (mercaptopyruvate sulfurtransferase and thiosulfate sulfurtransferase) rule out their involvement in SQOR activation. A combination of pharmacological inhibition and cystine supplementation studies points to the role of HS rather than low molecular weight persulfides in regulating SQOR. We posit that the solvent accessibility and reactivity of the trisulfide make SQOR vulnerable to reversible inhibition. Our study supports a model for trisulfide installation and activation via cysteine oxidation and sulfide addition and reveals a heretofore unrecognized mechanism for autoregulating SQOR by HS on demand.