We figured out the BH4 deficiency effect on heart mitochondria and cardio protection through 2DE proteomic analysis, mtDNA gene analysis, mitochondria oxygen consumption measurement tissue and cell microscopy observation combined with confocal microscopy with FACS study. Male 5weeks sepiapterin reductase double knock out mouse were used for BH4 deficient model vs wild type C57BL/6J mouse. Purely isolated heart mitochondria were subjected to 2DE proteomics and MALDI-MS analysis for revealing proteomic alteration between KO and wild type mouse mitochondria. The proteomic up-regulation of AIF protein and proline oxidase 2 coincided with the vulnerability and short life span of the BH4 deficient mouse. BH4 deficiency induced cell death and tissue damages were either shown in mtDNA fragmentation experiments and in situ cell death assay. The mitochondria functional alterations were detected by fluorescent dye associated mitochondria inner membrane potential measurement under confocal microscopy. Those mitochondrial function alterations were supported by down-regulation of mitochondria function regulate proteins including UQCR, Co Q9 and catalase. These findings could lead new BH4 mediated mitochondrial targeted therapy in cardiac protection.