Aim To investigate the protective effects of berberine on viral myocarditis in mice; To study the effects of berberine on Toll-like receptor 4 (TLR4), nuclear factor-κB (NF-κB) and tumor necrosis factor α (TNF-α). Methods According to random number table, 40 BALB/c mice were divided into four groups:normal group, model group, low dose berberine treatment group and high dose berberin treatment group. Except the normal group, the mice of other groups were injected intraperitoneally with addicted concentric Coxsackie virus B3 to establish viral myocarditis model. Low dose berberine treatment group and high dose berberin treatment group were injected intraperitoneally with 50 mg/kg and 100 mg/kg respectively. The pathological changes of myocardial tissue were observed by HE staining. The serum levels of creatine kinase isoenzyme MB (CK-MB), cardiac troponin T (cTnT) and TNF-α were determined by enzyme-linked immunosorbent assay kits. Western blot and real-time fluorescence quantitative PCR were used to detect the protein expression and mRNA expression of TLR4 and NF-κB in cardiac tissue. Results Compared with the model group, inflammatory cell infiltration and cell necrosis degree in myocardial tissues were significantly decreased, and the serum levels of CK-MB, cTnT and TNF-α were significantly reduced in the berberine treatment group. At the same time, the protein expression and mRNA expression of TLR4 and NF-κB were significantly down-regulated in myocardial tissue in the berberine treatment group. Conclusion Berberine can regulate the TLR4/NF-κB signal pathway, so as to protect the myocardial tissue of mice with viral myocarditis.