Metformin is a widely used anti-diabetic drug and increasing evidence suggests that metformin have profound cardioprotective effects under both diabetic and non-diabetic situations. The protective benefits of metformin have been proved in diabetic patients with cardiovascular complications and in experimental animals with myocardial infarction and cardiac hypertrophy. In the present study, we found that treatment with metformin inhibited the cardiac expression of pro-inflammatory cytokines including tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1β) and interleukin 6 (IL-6) in endotoxin-challenged mice. Treatment with metformin also alleviated the histological abnormalities in the heart, suppressed the upregulation of myeloperoxidase (MPO), decreased the elevation of creatinine kinase-myocardial band (CK-MB) and brain natriuretic peptide (BNP). Treatment with metformin promoted the phosphorylation of the catalytic α subunit of adenosine 5′-monophosphate-activated protein kinase (AMPKα), co-administration of AMPK inhibitor suppressed the stimulatory effects of metformin on AMPKα phosphorylation. Meanwhile, the suppressive effects of metformin on MPO, TNF-α, CK-MB and BNP were reversed by the AMPK inhibitor. On the contrary, administration of AMPK activator mimicked the effects of metformin on AMPKα phosphorylation, MPO upregulation, CK-MB release and BNP elevation. These evidence suggested that metformin might provide beneficial effects in endotoxin-induced acute myocarditis via activating AMPK-dependent anti-inflammatory mechanism.