Doxorubicin (DOX), a chemotherapeutic drug widely used in the clinical setting, is known to cause serious cardiotoxicity and greatly reduces the survival rate as well as quality of life of patients receiving chemotherapy. Peroxisome proliferation activated receptor alpha (PPAR alpha) is a type of ligand activated receptor of the nuclear hormone receptor family that regulates multiple gene expression. Several studies have shown that PPAR alpha has anti-apoptotic and cardio-protective effects. However, its role in DOX-induced cardiotoxicity is rarely reported. In this study, we observed decreased expression of PPAR alpha in the heart of tumor-bearing mice already treated with DOX; however, no such phenomenon was observed in tumor tissues. Next, we observed that the PPAR alpha agonist, fenofibrate (FENO), had no effect on tumor progression; however, it enhanced cardiac function in tumor-bearing mice treated with DOX. Subsequently, recombinant adeno-associated virus serotype 9 (rAAV9) was used to manipulate the expression of PPAR alpha in the heart of DOX-induced mice. Our results showed that PPAR alpha gene delivery reduced cardiac dysfunction and mitochondria-dependent apoptosis in DOX-induced mice. Furthermore, we found that PPAR alpha directly regulated the expression of mesenchyme homeobox 1 (MEOX1). Most importantly, the cardioprotective effects of PPAR alpha could be neutralized by knocking down MEOX1. In summary, PPAR alpha plays a vital role in DOX-induced cardiotoxicity and is a promising treatment target.