This work illustrated the mechanism contributing to the process of Phosphatidylinostiol 3-kinase (PI3K)/protein kinase B (PKB)/mammalian target of rapamycin (mTOR) signaling pathway, which has been demonstrated to play an important role in virus-induced apoptosis, which contributes to the Viral Myocarditis (VMC) pathogeneses. We examined the expression of Bax, Bim, caspase-3, caspase-9, and viral replication after Coxsackievirus B3 (CVB3) infection using the mTOR inhibitor and PI3K inhibitor pretreated HeLa cells, respectively. Apoptosis in different groups was determined by flow cytometry. Bax, Bim, caspase-9, and caspase-3 were examined by semiquantitative polymerase chain reaction (PCR) and Western blot analysis. The expression of CVB3 mRNA and viral capsid protein VP1 were analyzed by semiquantitative PCR and Western blot analysis distinctively. We found that rapamycin and LY294002 promote CVB3-induced cytopathic effect (CPE) and apoptosis. CVB3 replication in host cells is mediated in mRNA and protein expression by rapamycin and LY294002. Moreover, comparing with controls, at 12 and 24 h of postinfection (p.i.), Bim and Bax expression increased in cells after treated with rapamycin or LY294002, which also stimulates the activation of procaspase-9, and the CVB3-induced caspase-3 self-cleavage. However, in the meantime, the mRNA expression of caspase-9 and caspase-3 did not have an obvious change. In summary, our results demonstrated that the mTOR-signaling pathway plays an important role in CVB3-induced CPE and apoptosis, which is indispensable in VMC, via regulating Bim, Bax, caspase-9, caspase-3, and viral replication. Our findings may provide a new perspective and a deeper understanding of the mechanism of CVB3-induced apoptosis which, in turn, may help with the development of new therapy for the CVB3 infection.