Ischemic preconditioning (IPC), brief episodes of ischemia and reperfusion, is known to activate endogenous protective mechanisms against ischemia-reperfusion (IR) injury. Despite extensive studies, the role of hydrogen sulfide (H2S) produced in mitochondria by 3-mercaptopyruvate sulfurtransferase (3MPST) in IPC remains unknown. The aim of this study was to investigate the effect of o-carboxymethylhydroxylamine (OCA), a mitochondrial H2S synthesis inhibitor, on recovery of cardiac function and mitochondrial membrane integrity after ex vivo IPC and IR. The experiment was performed on 44 male Wistar rats divided into four groups: control; IR; IPC + IR; and OCA (50 mg/kg, i.p.) + IPC + IR. After recording cardiac function using the Langendorff apparatus, cardiac mitochondria were isolated and subjected to a swelling test. Our results demonstrated that OCA reduced left ventricular developed pressure, dP/dtmin, and coronary flow, and simultaneously increased the oxygen cost of myocardial work. Moreover, OCA abolished the cardioprotective effect of IPC, impairing the recovery of contractile activity and enhancing inefficient oxygen utilization in the ischemic myocardium. IR mitochondria showed a 1.54-fold increase in swelling amplitude in Ca2+-free medium, which was reduced by 15% in the IPC group. OCA increased the swelling amplitude 1.6-fold compared to IPC (even in Ca2+-free medium) and 1.36-fold compared to IR. Notably, mitochondria from the OCA-treated group were insensitive to Ca2+ at all tested concentrations (10−4-10−6 mol/l). These findings suggest that inhibition of mitochondrial H2S production impairs IPC mechanisms responsible for preservation of energy production and proper Ca2+ handling during reperfusion. Thus, H2S generated by 3MPST appears to play a crucial role in maintaining mitochondrial stability and mediating IPC-induced cardioprotection against IR injury. These results may have potential implications for pharmacological targeting of H2S signaling pathways.