UNLABELLED: Elevated plasma homocysteine levels are considered an independent risk factor for cardiovascular diseases. Experimental evidence has shown that hydrogen sulfide anion (HS(-)) protects the myocardium from ischemia/reperfusion (IR) injury. Both homocysteine levels and endogenous HS(-) production are mainly regulated by two transsulfuration enzymes, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CTH). We hypothesized that the transsulfuration pathway plays essential roles in the development of cardiac adaptive responses against ischemia, and investigated the roles of homocysteine, HS(-), and transsulfuration enzymes in fasting-induced cardioprotection against IR injury utilizing hyperhomocysteinemic Cbs (-/-) and Cth (-/-) mice. Langendorff-perfused hearts were subjected to 25-min global ischemia, followed by 60-min reperfusion. Two-day fasting ameliorated left ventricular dysfunction after reperfusion via propargylglycine- and glibenclamide-sensitive pathways in wild-type mice but not in Cbs (-/-) or Cth (-/-) mice, although fasting induced cardiac expression of several Nrf2 target antioxidant genes in both wild-type and Cth (-/-) mice. Intraperitoneal administration of sodium hydrosulfide (a HS(-) donor) at 24 h prior to IR improved myocardial recovery in wild-type mice but not in Cth (-/-) or high-methionine-diet-fed (thus intermediately hyperhomocysteinemic) wild-type mice. Quantitative analysis of reactive sulfur species using monobromobimane derivatization methods revealed that homocysteine efficiently captures HS(-) to form homocysteine persulfide in the hearts as well as in the in vitro reactions. Here we propose a novel molecular and pathophysiological basis for hyperhomocysteinemia; excessive circulatory homocysteine interferes with HS(-)-related cardioprotection against IR injury by capturing endogenous HS(-) to form homocysteine persulfide. KEY MESSAGE: Two-day fasting of mice ameliorates ischemia/reperfusion injury in Langendorff hearts. H2S-producing enzymes, CBS and CTH, are essential in fasting-induced cardioprotection. Administration of a H2S donor (NaHS) confers cardioprotection against IR injury. NaHS effects are absent in Cth (-/-), Cbs (-/-), and dietary hyperhomocysteinemic mice. Homocysteine captures cardioprotective HS(-) to form homocysteine persulfide.