Objective: To investigate the effects of interleukin-39 (IL-39) on mitophagy and myocardial injury in septic rats, as well as the regulatory role of Prohibitin2/PTEN induced putative kinase1/Parkin (PHB2/PINK1/Parkin) pathway. Methods: 80 rats were randomly divided into sham group, cecal ligation and puncture (CLP) group, CLP+IL-39 group, CLP+IL-39 inhibitor group, CLP+Ad-NC group, CLP+Ad-PHB2 group, CLP+IL-39+Ad-NC group, CLP+IL-39+Ad-PHB2 group, with 10 rats in each group. A rat sepsis model was established using CLP method. After corresponding intervention, echocardiography was used to detect the cardiac function of rats, ELISA was used to detect serum myocardial injury marker [cardiac troponin I (c-TnI), creatine kinase-myocardial band (CK-MB), lactate dehydrogenase (LDH)] and inflammatory factor [tumor necrosis factor (TNF-α), IL-1β, IL-6] levels. HE staining was used to observe myocardial pathological damage, transmission electron microscopy was used to observe myocardial mitochondrial morphology and mitophagosome formation, Western blotting was used to detect the expression of IL-39, PHB2, PINK1, Parkin, microtubule-associated protein light chain 3 Ⅱ (LC3 Ⅱ), and p62 protein in myocardium. Results: Compared with sham group, left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) values of rats in CLP group decreased, left ventricular end-systolic diameter (LVESD) and left ventricular end-diastolic diameter (LVEDD) values increased, serum c-TnI, CK-MB, LDH, TNF-α, IL-1β, and IL-6 levels increased. The myocardial cells were disordered with marked inflammatory cell infiltration, myocardial mitochondria were swollen and deformed, with cristae rupture and lysis, and mitophagosome formation was decreased, the expression of IL-39 and p62 in myocardium increased, while the expression of PHB2, PINK1, Parkin, and LC3 Ⅱ decreased (P<0.05). Compared with CLP group, LVEF and LVFS values of rats in CLP+IL-39 group decreased, LVESD and LVEDD values increased, serum c-TnI, CK-MB, LDH, TNF-α, IL-1β, and IL-6 levels increased. The myocardial cells were disordered with marked inflammatory cell infiltration, myocardial mitochondria were swollen and deformed, with cristae rupture and lysis, and mitophagosome formation was decreased, the expression of IL-39 and p62 in myocardium increased, while the expression of PHB2, PINK1, Parkin, and LC3 Ⅱ decreased (P <0.05). LVEF and LVFS values of rats in CLP+IL-39 inhibitor group increased, LVESD and LVEDD values decreased, serum c-TnI, CK-MB, LDH, TNF-α, IL-1β, and IL-6 levels decreased. Myocardial pathological damage was alleviated, mitochondrial structural damage was relieved, and mitophagosome formation was increased, the expression of myocardial IL-39 and p62 decreased, while the expression of PHB2, PINK1, Parkin, and LC3 Ⅱ increased (P <0.05). Compared with CLP+Ad-NC group, LVEF and LVFS values of rats in CLP+Ad-PHB2 group increased, LVESD and LVEDD values decreased, serum c-TnI, CK-MB, LDH, TNF-α, IL-1β, and IL-6 levels decreased. Myocardial pathological damage was alleviated, mitochondrial structural damage was relieved, and mitophagosome formation was increased, the expression of myocardial p62 decreased, while the expression of PHB2, PINK1, Parkin, and LC3 Ⅱ increased(P<0.05). Compared with CLP+IL-39+Ad-NC group, LVEF and LVFS values of rats in CLP+IL-39+Ad-PHB2 group increased, LVESD and LVEDD values decreased, serum c-TnI, CK-MB, LDH, TNF-α, IL-1β, and IL-6 levels decreased. Myocardial pathological damage was alleviated, mitochondrial structural damage was relieved, and mitophagosome formation was increased, the expression of myocardial p62 decreased, while the expression of PHB2, PINK1, Parkin, and LC3Ⅱincreased(P<0.05). Conclusion: IL-39 exacerbates myocardial injury in septic rats by inhibiting PHB2/PINK1/Parkin pathway mediated mitophagy.