The role of proton (H⁺) production from glucose metabolism in the recovery of myocardial function during post-ischemic reperfusion, and its alteration by insulin and other metabolic modulators were examined. Rat hearts were perfused in vitro with Krebs-Henseleit solution containing palmitate (1.2 mmol.L^-1) and glucose (11 mmol.L^-1) under non-ischemic conditions or during reperfusion following no-flow ischemia. Perfusate contained normal (n-INS, 50 mU.L^-1), zero (0-INS) or supplemental insulin (s-INS, 1000 mU.L^-1) or other metabolic modulators (dichloroacetate, DCA, 3 mmol.L^-1; oxfenicine, OXF, 1 mmol.L^-1; N6-cyclohexyladenosine, CHA, 0.5 µmol.L^-1). Relative to n-INS, 0-INS depressed rates of glycolysis and glucose oxidation in non-ischemic hearts and impaired recovery of post-ischemic function. Relative to n-INS, s-INS did not affect aerobic glucose metabolism, and did not improve recovery when present during reperfusion. When present during both ischemia and reperfusion, s-INS worsened recovery. Combinations of metabolic modulators with s-INS stimulated glucose oxidation ~2.5-fold in non-ischemic hearts and reduced H⁺ production. DCA and CHA, in combination with s-INS, improved recovery of function, but the addition of OXF to this combination provided no further benefit. Although DCA or CHA were each partially protective in hearts perfused with n-INS, optimal protection was achieved with a DCA+CHA combination; recovery of function was inversely proportional to H⁺ production during reperfusion. While supplemental insulin is not beneficial, elimination of the production H⁺ from glucose metabolism by simultaneous inhibition of glycolysis and stimulation of glucose oxidation optimizes recovery of post-ischemic mechanical function.