〔Abstract〕 Objective To investigate the possible mechanism of metformin(Met)-induced cardiomyocyte autophagy in protecting myocardial injury in septic mice. Methods The model of myocardial injury in septic mice was established by cecal ligation and puncture(CLP). Sixty Kunming mice were randomly divided into sham operation group(Sham group), model group(CLP group), model+dimethyl sulfoxide(DMSO) group(CLP+DMSO group), model+metformin(Met) group(Met group), model+Met+3-methyladenine(3-MA) group(Met+3-MA group), model+Met+compound C(CC) group(Met+CC group), with 10 mice in each group. The Met, Met+3-MA and Met+CC groups were intraperitoneally injected with Met(200 mg/kg) once a day for 2 weeks before modeling. The Met+3-MA group was intraperitoneally injected with 3-MA(10 mg/kg) 1 h before surgery. The Met+CC group was intraperitoneally injected with CC(20 mg/kg) 30 min before surgery. The model was established 24 h after the last injection of Met. The heart and blood of all mice were collected 24 h after surgery. The Western blot technique was employed to assess the relative expression levels of microtubule-associated protein 1 light chain 3(LC3) isoforms, namely LC3 I and LC3 II, autophagy effector protein 1(Beclin-1), ubiquitin-binding protein 62(p62), B-cell lymphoma/leukemia-2(Bcl-2), Bcl-2-associated X protein(Bax), adenosine monophosphate(AMP) kinase(AMPK) and phosphorylated AMPK(p-AMPK). Myocardial pathological changes were observed by hematoxylin-eosin(HE) staining. The changes of myocardial mitochondria and autophagosomes were observed by electron microscopy. Hematoxylin-eosin(HE) staining was used to observe the pathological changes of myocardium. Electron microscopy was used to observe the changes of myocardial mitochondria and autophagosomes. Results Compared with Sham group, the relative protein expression of Beclin-1, p62, p-AMPK/AMPK and LC3 II/LC3 I in CLP and CLP+DMSO groups had no statistical significance, but Bax increased and Bcl-2 decreased in CLP group(P<0.01). Compared with CLP group, the relative expression of Beclin-1 protein and LC3 II/LC3 I in Met group increased and p62 decreased(P<0.01), Bax decreased and Bcl-2 increased(P<0.01). Compared with Met group, the relative protein expression of Beclin-1 and LC3 II/LC3 I in Met+3-MA group decreased and p62 increased(P<0.05), Bax increased and Bcl-2 decreased(P<0.05). Besides, the relative protein expression of p-AMPK/AMPK in Met+CC group decreased(P<0.05). HE staining showed that there was no disorder in myocardial fibers in Sham group, and a large number of inflammatory cells infiltrated the myocardial fibers of CLP group in a clear disorder. The Met group showed vacuolar changes in the myocardium, while the Met+3-MA group showed disordered arrangement of myocardial fibers and a small amount of inflammatory cell infiltration. Under electron microscopy, the morphology of myocardial mitochondria in the Sham group was normal, while in the CLP group, the arrangement of mitochondrial cristae was disordered with vacuolar changes, and occasional autophagosomes were observed. Mitochondria in Met group showed slight swelling and a large number of autophagosomes. The mitochondria in the Met+3-MA group showed significant swelling with a small amount of autophagosomes. Conclusion The protective effect of metformin on myocardial injury in septic mice can reduce cardiomyocyte apoptosis and improve mitochondrial damage by activating AMPK signaling pathway to induce autophagy.