A high‐fat diet (HFD) has been associated with heart failure and arrhythmias; however, the molecular mechanisms underlying these associations are poorly understood. The mitochondria play an essential role in optimal heart performance, most of the energy for which is obtained from the oxidation of fatty acids. As such, chronic exposure to excess fatty acids may cause mitochondrial dysfunction and heart failure. To investigate the effects of a HFD on the mitochondrial function in the myocardium, 40 male rats were randomly divided into two groups and fed with either a normal diet or a HFD for 28 weeks. The myocardial lipid content, cardiac parameters and function, and mitochondrial morphology and function were evaluated. The expression of a number of genes involved in mitochondrial dynamics was measured using quantitative polymerase chain reaction and Western blot analyses. Proteomic analysis was also performed to identify the proteins affected by HFD treatment. Significant fat deposition in the myocardia, cardiac hypertrophy, and cardiac dysfunction were all observed in HFD‐treated rats. Electron microscopy showed abnormal mitochondrial density and morphology. In addition, abnormal expression of genes involved in mitochondrial dynamics, decreased mitochondrial DNA copy numbers, reduced complex I‐III and citrate synthase activities, and decreased mitochondrial respiration were observed in HFD‐treated rats. High performance liquid chromatography showed downregulated adenosine triphosphate (ATP) and adenosine diphosphate levels and an increased adenosine monophosphate (AMP)/ATP ratio. Proteomic analysis confirmed the alteration of mitochondrial function and impaired expression of proteins involved in mitochondrial dynamics in HFD‐treated rats. Mitochondrial dysfunction and impaired mitochondrial dynamics play an important role in heart dysfunction induced by a HFD, thus presenting a potential therapeutic target for the treatment of heart disease.