Tumor suppressor p53 is critical for suppressing all types of human cancers, including breast cancer. The p53 gene is somatically mutated in over half of all human cancers. The majority of the p53 mutations are missense mutations, leading to the expression of the full-length p53 mutants. Several hotspot mutations, including R175H, are frequently detected in human breast cancer. P53 cancer mutants not only lose tumor suppression activity but, more problematically, also gain new oncogenic activities. Despite correlation of the expression of p53 cancer mutants and the poor prognosis of human breast cancer patients, the roles of p53 cancer mutants in promoting breast cancer remain unclear. We used the humanized p53 cancer mutant knock-in (R175H) mice and mouse mammary tumor virus (MMTV)-Wnt-1 transgenic (mWnt-1) mice to specifically address the gain of function of R175H in promoting breast cancer. Although both R175H/R175HmWnt-1(R175HmWnt-1) and p53(-/-)mWnt-1 mice died from mammary tumor at the same kinetics, which was much earlier than mWnt-1 mice, most of the R175HmWnt-1 mice developed multiple mammary tumors per mouse, whereas p53(-/-)mWnt-1 and mWnt-1 mice mostly developed one tumor per mouse. The multiple mammary tumors arose in the same R175HmWnt-1 mouse exhibited different histological characters. Moreover, R175H gain-of-function mutant expands the mammary epithelial stem cells (MESCs) that give rise to the mammary tumors. As ATM suppresses the expansion of MESCs, the inactivation of ATM by R175H in mammary epithelial cells (MECs) could contribute to the expansion of MESCs in R175HmWnt-1 mice. These findings provide the basis for R175H to promote the initiation of breast cancer by expanding MESCs.