The kinase deficient erbB3 receptor frequently co-expresses and interacts with erbB2 in human breast cancer to activate the oncogenic signaling pathways, and thus promote breast cancer cell survival/proliferation. In the current study, we discovered that the expression of endogenous mouse erbB3 was increased in the mammary tumors-derived from wild type (wt) rat erbB2/neu-transgenic mice, and the co-expression of erbB2 and erbB3 significantly promoted mammary tumor proliferation in vivo. Co-immunoprecipitation assays detected a heterodimeric complex consisting of the transgene encoded protein rat erbB2 and the endogenous mouse erbB3 in the mammary tumors. Specific knockdown of mouse erbB3 dramatically inhibited proliferation of the mammary tumor cell lines-derived from the transgenic mice. Elevated expression of erbB3 protein, but not mRNA, was abserved in human breast cancer cells upon ectopic expression of erbB2. Additional studies revealed that overexpression of erbB2 downregulated three erbB3-targeting miRNAs, miR-125a, miR-125b, and miR-205, whereas the erbB2 kinase inhibitor (lapatinib) significantly enhanced expression of the three miRNAs in breast cancer cells, suggesting that erbB2 might regulate erbB3 expression through a miRNA-dependent mechanism. Furthermore, an anti-erbB3 monoclonal IgG1 antibody (Ab) in combination with Herceptin mainly inactivated Akt and significantly inhibited proliferation of erbB2-overexpressing breast cancer cells. Collectively, our data indicate that increased expression of erbB3 plays a pivotal role in activating downstream PI-3K/Akt pathway and promoting erbB2-driven mammary/breast tumorigenesis. Simultaneous targeting of erbB2 and erbB3 with two IgG1 Abs may be an effective strategy to treat breast cancer patients whose tumors overexpress both erbB2 and erbB3.