Abstract: Transmembrane protein 66 play important roles in cell apoptosis and tumorigenesis. In order to investigate the function of TMEM66 gene, the transgenic mouse model containing the mutant TMEM66 gene was constructed in this study. The transgenic mice were generated by using microinjection method and 312 injected zygotes were implanted into 13 foster mothers. The positive transgenic mice were confirmed by PCR and Southern blotting. Also, the stability of the transgene was confirmed by passage and the way of integration of the transgene was studied using inverse PCR method. In this study, 6 positive individuals among the 55 neonatal pups were found by Southern blotting. Three positive mice were found to be able to transfer the transgene to next generation. This result indicted that the transgene was integrated into host genome stably. Inverse PCR results indicated that the transgenic DNA fragment was integrated into the host genome in a serial multicopy manner. Therefore, the transgenic mice model containing the mutant TMEM66 gene was constructed successfully. This study offered useful model for further investigation of the function of TMEM66 gene.