Hyperhomocysteinemia (HHcy) is a risk factor for cardiovascular disease and has a strong correlation with heart failure. However, the effects of HHcy on cardiac tissue remain less well understood. To elucidate the role of p53-dependent apoptosis in HHcy-induced cardiac injury, we fed ApoE(-/-) mice with high methionine diet to establish HHcy model. Serum Hcy, cardiac enzymes, and lipids were measured. The protein levels of Noxa, DNMT1, caspases-3/9, and p53 were determined by enzyme-linked immunosorbent assay. Bcl-2 and Bax proteins were detected by immunohistochemistry staining. S-adenosyl methionine and S-adenosyl homocysteine concentrations were determined by high-performance liquid chromatography. The mRNA levels of p53 and DNMT1 were analyzed by real-time polymerase chain reaction (PCR) and the methylation levels of p53 were analyzed by nested methylation-specific-PCR. Our data showed that the concentrations of serum Hcy and lipids were increased in Meth group compared with the N-control group, which indicated that the model was established successfully. The expression levels of p53 and Noxa were increased in Meth group, while the methylation status of p53 was hypomethylation. The activities of caspase-3/9 were increased in Meth group compared with the N-control group. In addition, immunohistochemistry staining showed that the expression of Bax was significantly increased in Meth and Meth-F group compared with the N-control group. In summary, HHcy induces cardiac injury by up-regulation of p53-dependent pro-apoptotic related genes Noxa and Bax, while p53 DNA hypomethylation is a key molecular mechanism in pathological process induced by HHcy.