Background Caveolin-1 (Cav1)^−/− mice display impaired development of left ventricular pressure and increased left ventricular wall thickness but no dilated ventricle; these are typical findings in patients with heart failure with preserved ejection fraction (HfpEF). Aiming to clarify if dysfunctional endothelial nitric oxide synthase (eNOS) influences cardiomyocyte contractility, cardiac conduction system, or afterload/vascular resistance, we studied Cav1^−/−/eNOS^−/− mice. Methods Cardiac function was assessed in vivo by pressure-volume-catheterization of the left ventricle, echocardiography and electrocardiography. In addition, isolated tissue experiments were performed to evaluate cardiomyocyte contractility (atria) and vessel morphology and function (aorta). Histology, immunoblotting and quantitative polymerase chain reaction were applied to characterise radical formation and oxidative stress in the heart. Results Cardiac hypertrophy was completely reversed in Cav1^−/−/eNOS^−/− mice. The impaired pump function in Cav1^−/− mice was significantly improved in Cav1^−/−/eNOS^−/− mice, but no complete alignment with eNOS^−/− controls was achieved, indicating an additional eNOS-independent mechanism contributing to HFpEF in Cav1^−/− mice. It is unlikely that frequently occurring arrhythmias contributed to HFpEF in Cav1^−/− mice. In contrast, numerous eNOS-dependent and eNOS-independent vascular abnomalities could explain the cardiac phenotypes of Cav1^−/− mice. Conclusions Synergistic effects between eNOS-related cardiac hypertrophy and vascular hypercontractility appear to underlie the left ventricular dysfunction in Cav1^−/−mice. These findings provide insights relevant to the poorly understood pathophysiology of HFpEF.