The breast cancer-associated gene Di12 encodes a novel protein, which was found overexpressed in invasive ductal carcinomas of the breast. In experiments designed to assess the role of the Di12 gene in oncogenesis, the overexpression of 339 N-terminal amino acids of this gene in NIH3T3 cells resulted in cellular transformation and in vivo tumorigenesis. NIH3T3-Di12 tumor cell growth was partly reversible upon Di12 antisense treatment. In addition, transformation of the ER+ human breast cancer cell line MCF-7 resulted in hormone independent growth of these tumors in nude mice. Di12 expression in NIH3T3 and MCF-7 tumor cells was confirmed by RT-PCR and mabDi12 immunostaining. Immunohistochemistry using mabDi12 on an arrayed collection of 106 invasive breast tumors further underlined the expression of the gene in over 75% of advanced stage breast cancers. Our data indicate that Di12 expression is oncogenic in in vitro transformation and in vivo tumorigenic assays.