Paeonol and danshensu is the representative active ingredient of traditional Chinese medicinal herbs Cortex Moutan and Radix Salviae Milthiorrhizae, respectively. Paeonol and danshensu combination (PDSS) has putative cardioprotective effects in treating ischemic heart disease (IHD). However, the evidence for the protective effect is scarce and the pharmacological mechanisms of the combination remain unclear. The present study was designed to investigate the protective effect of PDSS on isoproterenol (ISO)-induced myocardial infarction in rats and to elucidate the potential mechanism. Assays of creatine kinase-MB, cardiac troponin I and T and histopathological analysis revealed PDSS significantly prevented myocardial injury induced by ISO. The ISO-induced profound elevation of oxidative stress was also suppressed by PDSS. TUNEL and caspase-3 activity assay showed that PDSS significantly inhibited apoptosis in myocardia. In exploring the underlying mechanisms of PDSS, we found PDSS enhanced the nuclear translocation of Nrf2 in myocardial injured rats. Furthermore, PDSS increased phosphorylated PI3K and Akt, which may in turn activate antioxidative and antiapoptotic signaling events in rat. These present findings demonstrated that PDSS exerts significant cardioprotective effects against ISO-induced myocardial infarction in rats. The protective effect is, at least partly, via activation of Nrf2/HO-1 signaling and involvement of the PI3K/Akt cell survival signaling pathway.