Accumulating evidences have demonstrated that overwhelming inflammation occurs in the process of Coxsackievirus B3 (CVB3)-induced acute viral myocarditis (AVM). No specific therapy is available. More than an effective Janus-associated kinase (JAK) inhibiter, ruxolitinib exerts a critical role in the inflammatory diseases. In this study, we investigated the potential effect of ruxolitinib on CVB3-induced acute viral myocarditis.In vivo, BALB/c mice were intraperitoneally injected of CVB3, treated of a successive gavage of ruxolitinib for seven days, and subjected to a series of analysis. In vitro, primary bone marrow-derived macrophages (BMDMs) and cardiac fibroblasts were isolated, cultured, treated, harvested and finally detected.In vivo, acute viral myocarditis was successfully induced by the injection of CVB3 characterized by impaired cardiac function, predominant infiltration of inflammatory cells, necroptosis of myocardium, great increase of cardiac troponin I (cTnI) and cytokine levels, replication of CVB3, and excessive activation of JAK-STAT pathways. Oral administration of ruxolitinib suppressed the activation of JAK-STAT pathway in a dosage-dependent way, lessened the infiltration of inflammatory cells and necroptosis of myocardium, reduced the levels of cTnI and cytokines, and finally alleviated CVB3-induced cardiac dysfunction, with the reduced production of type I interferon and no promising effect on the replication of CVB3. In vitro, the treatment of ruxolitinib inhibited the activation of JAK-STAT pathway and increase of multiple cytokines mRNA levels in BMDMs and had no protective effect against CVB3 replication in cardiac fibroblasts.Our study suggested that ruxolitinib ameliorated CVB3-induced AVM by inhibiting the activation of JAK-STAT pathway, infiltration of inflammatory cells and necroptosis of myocardium, which may provide a novel strategy for AVM therapy.Copyright © 2023. Published by Elsevier B.V.