This study tested if the protective effect of quercetin (QUR) against experimentally-induced acute myocardial infarction (AMI) in rats involves modulating the Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) pathway. Rats were divided into 6 groups as sham-operated (control), control + QUR, AMI, AMI + QUR, AMI + S3I-210 (a STAT3 inhibitor), and AMI + QUR + S31-201. QUR (50 mg/kg/orally) and S3I-201 (a STAT3 inhibitor) (5 mg/kg/i.p.) were administered for 7 days before the induction of AMI and the experiment was ended 24 h post-AMI. Pre-treatment with QUR reduced the infarct size, improved the left ventricular (LV) functions and the structure of the myofibrils and the mitochondria, and reduced circulatory levels of lactate dehydrogenase (LDH), creatinine-kinase MB (CKMB), and troponin-I. QUR also reduced LV levels of reactive oxygen species (ROS) and malondialdehyde (MDA), inhibited the opening of the mitochondria transition pores (mtPTP), and reduced protein levels of cytochrome-C, cleaved caspase-3 and p-JAK2 (Tyr1007/1008) in the LVs of AMI rats. In the LV of both the control and AMI rats, QUR didn't affect the levels of p-JAK2 but significantly increased the levels of total glutathione (GSH) and manganese superoxide dismutase (MnSOD), reduced the levels of Bax and the nuclear levels and activity of NF-κB p65, tumor necrosis-factors-α (TNF-α), interleukin-6 (IL-6), and p-STAT1 (Ser727) but further increased the levels of p-STAT3 (Ser727). All these effects exerted by QUR were partially reversed but the decrease in nuclear protein levels and activity of NFκB, levels of TNF-α and IL-6, and pSTAT3 were completely prevented by co-administration of S3I-201. In conclusion, QUR protects against MI by upregulation of antioxidants and activation of STAT3.