Phosphoinositide 3-kinase [PI3K (p110α)] is able to negatively regulate the diabetes-induced increase in NADPH oxidase in the heart. Patients affected by diabetes exhibit significant cardiovascular morbidity and mortality, at least in part due to a cardiomyopathy characterized by oxidative stress and left ventricular (LV) dysfunction. Thus, PI3K (p110α) may represent a novel approach to protect the heart from diabetes-induced cardiac oxidative stress and dysfunction. In the present study, we investigated the therapeutic potential of a delayed intervention with cardiac-targeted PI3K gene therapy, administered to mice with established diabetes-induced LV diastolic dysfunction. Diabetes was induced in 6-week-old male mice by streptozotocin (STZ). After 8 weeks of untreated diabetes, LV diastolic dysfunction was confirmed by a reduction in echocardiography-derived transmitral E/A ratio. Diabetic and non-diabetic mice were randomly allocated to receive either recombinant adeno-associated viral vector-6 carrying a constitutively-active PI3K construct (recombinant adeno-associated-virus 6-constitutively active PI3K (p110α) (caPI3K) (rAAV6-caPI3K), single i.v. injection, 2 × 1011 vector genomes) or null vector, and were followed for a further 6 or 8 weeks. At study endpoint, diabetes-induced LV dysfunction was significantly attenuated by a single administration of rAAV6-caPI3K, administered 8 weeks after the induction of diabetes. Diabetes-induced impairments in each of LV NADPH oxidase, endoplasmic reticulum (ER) stress, apoptosis, cardiac fibrosis and cardiomyocyte hypertrophy, in addition to LV systolic dysfunction, were attenuated by delayed intervention with rAAV6-caPI3K. Hence, our demonstration that cardiac-targeted PI3K (p110α) gene therapy limits diabetes-induced up-regulation of NADPH oxidase and cardiac remodelling suggests new insights into promising approaches for the treatment of diabetic cardiomyopathy, at a clinically relevant time point (after diastolic dysfunction is manifested).