BACKGROUND: Viral myocarditis (VMC) is an inflammatory myocardial condition triggered by viral infections which involves pathogenic-related damage and immune-mediated damage. However, the precise immunopathogenic mechanisms underlying VMC remain elusive. METHODS: We performed single-cell RNA sequencing on mouse hearts during the acute phase of CVB3-induced VMC. After manually annotating cell types, functional analyses of macrophage were performed by cell ratio changes, customized gene set module scoring and CellPhoneDB. Utilizing indirect co-culture experiments in vitro, the effects of macrophage-derived SPP1 on cardiac fibroblasts were investigated. Depletion of macrophages and inhibition of SPP1 expression in mice were carried out to study the effects of macrophage-derived SPP1 on cardiac function, inflammation levels, and myocardial injury in mice with VMC. RESULTS: Our data revealed that macrophages are the major immune cells which infiltrate the heart during the acute phase of VMC, particularly a macrophage subpopulation which highly expresses Spp1 (Spp1 CONCLUSION: Our findings suggested that Spp1