To explore the potential protective effect of Tanshinone IIA on myocardial cell apoptosis and elucidate the underlying molecular mechanisms. The rat heart cell H9c2 was treated by either H2O2 or doxorubicin (DOX) to mimic oxidative stress and DNA damage conditions in vivo. Cell growth was monitored by optical microscope observation or CCK-8 counting kit. The relative expression of miR-133 and U6 snoRNA was semi-quantitated by RT-PCR or real-time PCR. Cell apoptosis was analyzed by flow cytometry with Annexin V/PI double staining. The microRNA binding sites were predicted by online bioinformatics tools. The regulatory effect of miR-133 on caspase-9 was measured by luciferase reporter assay. Apoptosis pathway factors were analyzed by immunoblotting. Our data demonstrated that Tanshinone IIA significantly ameliorated myocardial apoptosis induced by either H2O2 or DOX. The protective effect was likely mediated by up-regulation of miR-133. We further identified Caspase-9 as the target of miR-133. Tanshinone IIA treatment significantly reversed down-regulation of miR-133 under harsh conditions and in turn suppressed evoking of Caspase-9 and related apoptotic effectors, which consequently contributed to the improvement of myocardial injury. In conclusion, Tanshinone IIA ameliorated myocardial apoptosis via restoration of miR-133 and suppression Caspase-9 signaling cascade, which underlies its well-proven clinical benefit and warrants larger scale clinical applications.