Many activators of K(ATP) channels exhibit cardioprotective effects, mainly mediated by channels expressed on mitochondria (mito-K(ATP)). Previous results showed anti-ischemic effects of the spiro-cyclic derivative A, on isolated rat hearts. In this work this molecule was more extensively studied and diazoxide was used as reference mito-K(ATP) opener. The studies were performed on an in vivo rat model of myocardial infarct and on heart-derived H9c2 cells exposed to an anoxic environment. The mechanism of action was further investigated on isolated rat heart mitochondria. In the model of myocardial infarct compound A and diazoxide produced significant cardioprotective effects, antagonised by the selective mito-K(ATP) blocker 5-hydroxydecanoic acid (5-HD). Compound A, like diazoxide, produced modest and non-significant hypotensive responses, while the hyperglycaemic effects of diazoxide were not observed for the new compound. Protective effects of compound A and diazoxide were also recorded in H9c2 cells and again were inhibited by 5-HD. Compound A and diazoxide caused swelling of cardiac mitochondria, in agreement with the profile of mito-K(ATP) openers. Both compounds evoked concentration-dependent Ca2+-release from Ca2+-preloaded mitochondria, prevented mitochondrial Ca2+-uptake and caused mitochondrial membrane depolarisation. These effects were antagonised by ATP, the endogenous K(ATP) inhibitor. In conclusion, compound A exhibits a promising profile of an anti-ischemic agent, with a mechanism likely to be linked to the activation of mito-K(ATP) channels, and, because of its chemical characteristics such as structural rigidity and chirality due to the spiro-cyclic moiety, represents an interesting template for development of analogues further improved in activity and selectivity.