Objective：This study aims to construct mitochondria-targeted ECSIT transgenic mice，identify the phenotype of the mice generated，and establish an animal model for ECSIT gene-related function. Methods：The expression vector pCAG-OTCL-ECSIT-3Xflag-BPA was constructed，and the linearized targeting vector was transferred into embryonic stem cells（ES cells）by electro transduction. The positive clones were injected into the blastocyst and transplanted into the surrogate mice to breed the chimeric mice. Chimeric mice and C57BL/6J mice then gave birth to potential heterozygote founders. The gene type of the mice was assayed by PCR. The cardiac function of mitochondria-targeted ECSIT mice was analyzed using small animal ultrasound. Results：Homologous recombinant vector pCAG-OTCL-ECSIT-3Xflag-BPA with mitochondrial targeted-overexpression of ECSIT was successfully constructed and identified correctly by PCR. Mitochondrial targeted-overexpression of ECSIT gene and blastocyst injection were completed and identified correctly by PCR. Mitochondrial and cytoplasmic proteins were isolated from transgenic mice，and ECSIT protein was specificly overexpressed in mitochondrion. Conclusion：Mitochondria-targeted ECSIT mice were successfully constructed；overexpression of ECSIT in mitochondrion had no significant effect on cardiac function in 8-week-age mice.