The mitochondrial permeability transition pore (mPTP) is widely accepted as an end-effector mechanism of conditioning protection against injurious ischaemia/reperfusion. However, death can be initiated in cells without pre-requisite mPTP opening, implicating alternate targets for ischaemia/reperfusion injury amelioration. Matrix metalloproteinases (MMP) are known to activate extrinsic apoptotic cascades and therefore we hypothesised that MMP activity represents an mPTP-independent target for augmented attenuation of ischaemia/reperfusion injury. In ex vivo and in vivo mouse hearts, we investigated whether the MMP inhibitor, ilomastat (0.25 μmol/l), administered upon reperfusion could engender protection in the absence of cyclophilin-D (CyPD), a modulator of mPTP opening, against injurious ischaemia/reperfusion. Ilomastat attenuated infarct size in wild-type (WT) animals [37 ± 2.8 to 22 ± 4.3 %, equivalent to ischaemic postconditioning (iPostC), used as positive control, 27 ± 2.1 %, p < 0.05]. Control CyPD knockout (KO) hearts had smaller infarcts than control WT (28 ± 4.2 %) and iPostC failed to confer additional protection, yet ilomastat significantly attenuated infarct size in KO hearts (11 ± 3.0 %, p < 0.001), and similar protection was also seen in isolated cardiomyocytes. Moreover, ilomastat, unlike the cyclophilin inhibitor cyclosporine-A, had no impact upon reactive oxygen species-mediated mPTP opening. While MMP inhibition was associated with increased Akt and ERK phosphorylation, neither Wortmannin nor PD98059 abrogated ilomastat-mediated protection. We demonstrate that MMP inhibition is cardioprotective, independent of Akt/ERK/CyPD/mPTP activity and is additive to the protection observed following inhibition of mPTP opening, indicative of a parallel pathway to protection in ischaemic/reperfused heart that may have clinical applicability in attenuating injury in acute coronary syndromes and deserve further investigation.