Mitochondrial dysfunction caused by mitochondrial permeability transition pore opening (mPTP) and excessive calcium accumulation is a major contributor to cell and tissue damage during myocardial infarction and ischaemia-reperfusion injury. Although the role of calcium in induction of the mPTP opening is established, exact molecular mechanism of this process is not understood. We hypothesise that toxic effect of calcium accumulation is mediated by its interaction with inorganic polyphosphate, a biological polymer made of 10 to 100 orthophosphates. To test this hypothesis we investigated kinetics of the mPTP opening in permeabilised and intact cardiac cells depleted of polyphosphate under conditions of calcium overload. Polyphosphate depletion was achieved by targeted expression of specific polyphosphate hydrolysing enzyme using viral infection system. mPTP kinetics were estimated from the rates of TMRM, calcein red and X-rhod-1 release from mitochondria using confocal fluorescent microscopy approach. We found that although depletion of mitochondria of cardiac cells of polyphosphate did not affect their ability to accumulate calcium, it significantly inhibited mPTP opening. Inhibitory effect was observed in polyphosphate-depleted in experiments when calcium was added directly to mitochondria of permeabilised cells (80% decrease in rate of calcein red release, n=27; p<0.01 and 70% decrease in rate of X-Rhod-1 release n=17, p<0.01) as well as in experiments with intact cells when calcium overload was induced by creating conditions mimicking ischaemia-reperfusion injury (55% decrease in rate of calcein red release, n=19; p<0.05). Our findings suggest that inorganic polyphosphate is previously unrecognised major activator of mPTP during conditions of ischaemia-reperfusion injury.