Cytosolic NADPH generated by the oxidative pentose phosphate pathway (oxPPP) is critically important for redox homeostasis and function of isolated cardiomyocytes. We tested whether knocking down glucose-6-phosphate dehydrogenase (G6PD), the first and rate-limiting enzyme in the oxPPP, impairs cardiac performance, in vivo. In 5 conscious, chronically instrumented dogs, hemodynamics and cardiac oxygen consumption were measured at baseline and during dobutamine stress. 1013 p.f.u. adenoviral vectors carrying DNA encoding for anti-G6PD short-interfering RNA (siRNA) were then delivered to the heart via coronary sinus. Eight days after adenovirus delivery, dogs underwent the same protocol described above, with an additional infusion of [U-13C]-glucose. 13C-NMR spectroscopy was performed in frozen myocardial tissue samples. siRNA administration: 1) did not significantly alter heart rate and blood pressure at baseline and in response to dobutamine; 2) depressed LV stroke work (baseline: 422.2±26.4 vs 783.6±181.4 mm*mmHg, P<0.05); 3) caused a decrease in MVO2/beat (baseline: 2.2±0.3 vs 3.6±0.5 ml/beat/100g, P<0.05). During dobutamine stress, the ratio stroke work/MVO2/beat, an index of mechanical efficiency, was significantly lower after siRNA administration compared to control (at 10 mg/kg/min dobutamine: 302.4±26.0 vs 228.8±27.2, P<0.05). In siRNA treated hearts, NMR analysis revealed a 28.8±4.1% reduction in 6-phospho-13C-gluconate, and a 32.2±2.7% reduction in 13C-ribose, two representative products of the oxPPP. No evidence of myocardial inflammation was found. Our results are the first to show that a partial interruption of the oxPPP impairs cardiac contractile performance and mechanical efficiency.