To investigate the effects of uncoupling protein 2 (UCP2) overexpression on mitochondrial dynamics (mitochondrial division and fusion) of sepsis myocardial injury in rats. Forty male Sprague-Dawley (SD) rats were randomly divided into four groups (n = 10): sham operation group (Sham group) using normal saline instead of transfection and simulating cecal ligation and perforation (CLP); CLP group using normal saline instead of transfection, performing CLP to induce sepsis; adeno-associated virus (AAV) group using CLP after myocardial transfection with empty virus; UCP2 overexpression group (UCP2 group) CLP was performed 3 weeks after AAV-UCP2 (1×1015 vg/L, a total of 60 μL) myocardial transfection. The rats in each group were examined by echocardiography at 24 hours after the CLP, and then the rats were sacrificed immediately to harvest myocardial tissue. Myocardial ultrastructural changes were observed under the electron microscope, the expression of regulatory proteins related to myocardial mitochondrial dynamics [optic atrophy 1 (Opa1), dynamin-related protein 1 (Drp1) and fission 1 (Fis1)] were detected by Western Blot, and the level of mitochondrial adenosine triphosphate (ATP) production was detected by chemiluminescence. (1) The echocardiographic results showed that there was no significant difference in left ventricular mass (LVM) and stroke volume (SV). Compared with Sham group, left ventricular diastolic anterior wall thickness (LVAWd), left ventricular systolic anterior wall thickness (LVAWs), left ventricular diastolic posterior wall thickness (LVPWd), left ventricular systolic posterior wall thickness (LVPWs), left ventricular ejection fraction (LVEF) and left ventricular short axis shortening rate (LVFS) were significantly increased in CLP group and AAV group, while left ventricular systolic diameter (LVEDs), left ventricular diastolic diameter (LVEDd), left ventricular end-systolic volume (LVESV), and left ventricular end-diastolic volume (LVEDV) were significantly decreased. Compared with CLP group and AAV group, LVAWs, LVEF, LVFS were significantly decreased in UCP2 group, and LVEDs, LVEDV and LVESV were significantly increased [LVAWs (mm): 3.82±0.42 vs. 4.34±0.30, 4.44±0.12; LVEF: 0.921±0.038 vs. 0.979±0.019, 0.991±0.010; LVFS: (65.33±6.56)% vs. (80.11±8.23)%, (85.31±6.11)%; LVEDs (mm): 1.81±0.36 vs. 0.89±0.54, 0.60±0.17; LVEDV (μL): 137.09±50.05 vs. 89.72±53.04, 85.42±40.99; LVESV (μL): 10.48±4.59 vs. 2.48±3.52, 2.58±2.50, all P < 0.05]. (2) Electron microscope showed that the structure of myocardial fibers in the Sham group was clear and aligned with complete intervertebral disc and mitochondrial structure, no damage to mitochondrial membranes, and tight arrangement of cristae. In CLP group and AAV group, muscle fiber breakage, sarcoplasmic reticulum expansion, severe mitochondrial swelling and even cristage structure disorder were observed. In the UCP2 group, only myocardial fiber edema was observed, and the muscle fiber structure was more complete than that of Sham group and AAV group. The mitochondria were slightly swollen and the cristae were intact. (3) Western Blot showed that there was no significant difference in the expression of Opa1 and Fis1 in the four groups. The expression of Drp1 in CLP group and AAV group were significantly higher than that in Sham group. The expression of Drp1 in UCP2 group was significantly lower than that in CLP group and AAV group (Drp1/β-actin: 1.01±0.03 vs. 1.39±0.03, 1.49±0.03, both P < 0.05). (4) The results of immunofluorescence showed that the ATP content of CLP group and AAV group were significantly lower than that of Sham group; the ATP content of UCP2 group was significantly higher than that of CLP group and AAV group (μmol/L: 1.99±0.15 vs. 1.10±0.17, 1.13±0.19, both P < 0.05). UCP2 overexpression can significantly improve the systemic systolic function of myocardium in sepsis rats, protect myocardial mitochondrial ultrastructure, inhibit mitochondrial division, and improve mitochondrial ATP synthesis.