DNA cytosine deaminase APOBEC3B (A3B) is one of the endogenous sources of somatic mutations in many types of human cancers and is associated with tumor progression rather than tumorigenesis. However, it remains uncertain whether APOBEC3B-induced mutations accelerate tumor progression or not. In this paper, we established a mouse model with A3B overexpression and investigated whether the introduction of A3B overexpression accelerates tumor development in Tp53 hemizygous mice. A3B expression was validated by qPCR, immunoblotting, and immunohistochemistry in mouse tissues, and in vitro CDA assays revealed that A3B has its CDA activity in mouse tissues. However, we did not observe any difference in tumor development between the mice with or without A3B expression. A3B expression and its CDA activity were confirmed in tumor tissues of mice overexpressing A3B. Therefore, we concluded that the introduction of A3B overexpression did not accelerate tumor development in Tp53 hemizygous mice. Our mouse model with A3B overexpression is well-validated and useful for further research.