The mitogen-activated protein kinase phosphatases (MKPs) are a family of dual-specificity phosphatases that inactivate MAPKs by dephosphorylation. Impairment of MKP-1 expression in insulin resistance has been suggested to affect the cardioprotective properties of insulin. We hypothesized that manipulation of its activity during myocardial ischaemia/reperfusion of control as well as insulin-resistant rats may affect the outcome. Hearts from 16week dietary induced obese Wistar rats and their age matched controls were isolated, perfused in the working mode and subjected to 15min global ischaemia / 30min reperfusion or 35min coronary artery ligation/ 60min reperfusion. Hearts received insulin (1mIU/ml), a MKP-1 inhibitor (sanguinarine 2.5uM), or insulin+sanguinarine for 15min pre- and 10min post-ischaemia. Endpoints were functional recovery and infarct size. Hearts from control and experimental groups were freeze-clamped either immediately after removal from the animal (baseline) or at 10min reperfusion after global ischaemia and Western blot analysis done for total and phosphorylated MKP-1. Insulin treatment significantly increased total work recovery while sanguinarine abolished the insulin-mediated protection. Insulin had no effect on infarct size while sanguinarine reduced infarct size. Insulin increased while sanguinarine attenuated phosphorylation of MKP-1 at 10min reperfusion. Inhibition of MKP-1 with sanguinarine abolished the insulin-induced improvement in functional recovery, but reduced infarct size. Although the data suggest a role for this phosphatase in insulin-induced cardioprotection, the multiple downstream effects of insulin hamper interpretation of the data obtained. In addition, the effects of sanguinarine per se in myocardial ischaemia/reperfusion need to be further elucidated.