Objectives: To investigate the effect of lupeol in a mouse model of viral myocarditis induced by coxsackie virus B3 (CVB3). Methods: Mice were separated into controls (DMEM, n ¼ 20) and CVB3 infected groups (i.e., untreated CVB3 [n ¼ 40]; CVB3 þ lupeol 50 mg/kg [n ¼ 40]; CVB3 þ lupeol 100 mg/kg [n ¼ 40]; CVB3 þ small interfering RNA (siRNA)-toll-like receptor 4 (TLR4) [n ¼ 20]; siRNA þ EXP-H mice [n ¼ 20]). Reverse transcription polymerase chain reaction (RT-PCR), western-blot assay, immunohistochemistry, enzyme-linked immunosorbent (ELISA) assay and histopathology were performed to investigate the cardioprotective role of lupeol. Results: The elevated pro-inflammatory cytokines in CVB3-infected mice (i.e., interleukin-1b [IL-1b]; interleukin-6 [IL-6]; tumour necrosis factor-a [TNF-a]) were significantly reduced by lupeol 50 or 100 mg/kg. Interestingly, the mRNA level and protein level of toll-like receptor 4 (TLR4) were inhibited by lupeol. Conclusions: Lupeol alleviates CVB3-induced viral myocarditis and myocardial damage in mice. The underlying mechanism may due to downregulation of TLR4.