Carnitine acts as a buffer of acetyl-CoA units in the mitochondria, as well as facilitating transport of fatty acids. Mildronate can block the biosynthesis of L-carnitine, reducing the uptake of fatty acids into the mitochondria by CPT-1. The purpose of this study was to investigate the effect of Mildronate treatment on cardiac function and metabolism in the healthy and diabetic rat heart. We showed that daily injections of Mildronate can alter cardiac metabolism in the in vivo diabetic and healthy rat heart, without any functional changes. However, when exposed to ischemia ex-vivo, Mildronate treated hearts improve their functional recovery post-ischemia. Such studies allow a better understanding of the interactions between metabolism and function in the diabetic heart and may provide new insight into novel therapeutics.