Objective To explore the effect of luteolin (Lu) on myocardial mitochondrial injury in rats with heart failure (HF) induced by isoprenaline. Methods Thirty male SD rats (6-7 weeks old) were randomly divided into control group, model group (HF) and Lu intervention group (HF+Lu), with 10 animals in each group. Rat model of HF were established by intra-peritoneal injection of 50mg/(kg·d) isoproterenol in HF and HF+Lu group, while the rats in control group were intraperitoneally injected with normal saline equally. At the same time, the rats in HF+Lu group were also given 3ml Lu 50mg/(kg·d) by gavage, while the rats in the other 2 groups were given 3ml 50g/L sodium carboxymethyl cellulose by gavage. After intervention for 10d, echocardiography was performed to detect cardiac function; on the eleventh day transmission electron microscopy was used to observe the ultrastructural changes of myocardial mitochondria; and fluorescent enzyme labeling was employed to measure mitochondrial membrane potential (MMP), and activities of succinate dehydrogenase [JP+2](SDH) and cytochrome C oxidase (COX); RT-PCR was applied to detect theexpression of apoptosis-related genes Bax, caspase3 and caspase9. SPSS statistics 19.0 was used to analyze the data. Results Echocardiography showed that compared with control group, left ventricular ejection fraction [LVEF, (68.0±3.1)% vs (86.0±4.5)%, P=0.023] and left ventricular fraction shortening [LVFS, (32.0±3.7)% vs (43.0±2.5)%, P=0.002] were decreased significantly, while left ventricular end-systolic diameter [LVEDs, (4.10±0.29) vs (3.20±0.27)mm, P=0.010] and left ventricular end-diastolic diameter [LVEDd, (7.10±0.34) vs (5.87±0.35)mm, P=0.034] were increased markedly in the HF group. After Lu intervention, LVEF [(78.0±5.8)% vs (68.0±3.1)%, P=0.028] and LVFS [(39.0±1.5)% vs (32.0±3.7)%, P=0.006] were elevated while LVEDs [(3.40±0.23) vs (4.10±0.29)mm, P=0.043] and LVEDd [(5.65±0.21) vs (7.10±0.34)mm, P=0.019] were decreased in the HF+Lu group compared with HF group. Under a transmission electron microscope, the myocardial mitochondria in control group had clear and complete structure, clear Z line, well-arranged thick and thin filaments, and mostly round or oval shaped. Myocardial mitochondria in HF group were swollen and uneven in size, with damaged membrane integrity, broken or dissolved cristae, and formed vacuoles. Compared with HF group, the damage of myocardial mitochondria in HF+Lu group was significantly reduced, cristae was clear, membrane integrity was better, and no vacuoles were found. Compared with control group, the MMP and activities of SDH and COX in HF group were significantly decreased (P<0.05), and the mRNA levels of Bax, caspase3 and caspase9 were significantly increased (P<0.05); compared with HF group, the MMP and activities of COX and SDH in HF+Lu group were significantly increased (P<0.05), while the levels of above genes were significantly decreased (P<0.05). Conclusion Lu plays a protective role in HF rats by reducing isoprenaline-induced myocardial mitochondrial damage and apoptosis.