Background Increased H2S availability reduces injury associated with myocardial ischemia (MI) and ischemia–reperfusion. The mitochondrial enzyme thiosulfate sulfurtransferase (TST) has a putative role in removal of H2S and is a potential target to increase H2S bioavailability. This study investigated the hypothesis that TST deletion (Tst−/−) would improve outcome following MI. Methods TST expression was assessed by qRT-PCR and Western blotting. Tst−/− and WT mice were examined in vivo by ultrasound. MI was induced by coronary artery ligation in vivo, or ex vivo inperfused hearts, when 30 min ischemia was followed by 120 min reperfusion. Results qRT-PCR and Western blots confirmed the presence of TST in the murine heart and deletion in Tst−/−. Tst−/− mice survived to adulthood and had normal cardiac function. Expression of the H2S synthesising enzyme, cystathionine gamma-lyase (CSE), was reduced in Tst−/− relative to WT hearts (n = 5, p Discussion TST is present in the heart and deletion does not influence function. Tst−/− mice have reduced cardiac CSE expression, suggesting reduced H2S synthetic capacity. This may underlie the increased susceptibility of Tst−/− mice to myocardial injury and increased mortality. Conclusion TST may act with CSE to regulate H2S availability. Alteration in the balance of these enzymes has no overt physiological effect, but is associated with reduced capacity of the heart to resist ischemic stress.