Sirtuins (Sirts) are implicated in regulating a myriad of biologic functions ranging from cell growth and metabolism to longevity. Here, we show that nuclear Sirt, Sirt6, and mitochondrial Sirt, Sirt3, regulate each other's activity and protect the heart from developing diabetic cardiomyopathy. We found that expression of both Sirt6 and Sirt3 was reduced in cardiomyocytes treated with palmitate and in hearts of mice fed with a high-fat, high-sucrose (HF-HS) diet to develop obesity and diabetes. Conversely, whole-body overexpressing Sirt6 transgenic (Tg.Sirt6) mice were protected from developing obesity and insulin resistance when fed with the same HF-HS diet. The hearts of Tg.Sirt6 mice were also protected from mitochondrial fragmentation and decline of Sirt3, resulting otherwise from HF-HS diet feeding. Mechanistic studies showed that Sirt3 preserves Sirt6 levels by reducing oxidative stress, whereas Sirt6 maintains Sirt3 levels by up-regulating nuclear respiratory factor 2 (Nrf2)-dependent Sirt3 gene transcription. We found that Sirt6 regulates Nrf2-mediated cardiac gene expression in 2 ways; first, Sirt6 suppresses expression of Kelch-like ECH-associated protein 1 (Keap1), a negative regulator of Nrf2, and second, Sirt6 binds to Nrf2 and antagonizes its interaction with Keap1, thereby stabilizing Nrf2 levels in cardiomyocytes. Together, these studies demonstrate that Sirt6 and Sirt3 maintain each other's activity and protect the heart from developing diabetic cardiomyopathy.-Kanwal, A., Pillai, V. B., Samant, S., Gupta, M., Gupta, M. P. The nuclear and mitochondrial sirtuins, Sirt6 and Sirt3, regulate each other's activity and protect the heart from developing obesity-mediated diabetic cardiomyopathy.