Recent studies have highlighted a role of HER3 in ER and HER2-driven breast cancers. We sought to investigate the role of patient-derived HER3 mutations in ER+ and HER2+ breast cancer cells using ectopic expression of HER3 mutants. We found that HER3T355I mutant is activating with increased cell proliferation in ER+ T47D and MCF-7 breast cancer cells lacking HER2 over-expression. Immunoblotting and receptor tyrosine kinase array results indicated that T47D and MCF-7 cells expressing HER3T355I had increased p-HER4 and p-HER1 expression. Our data showed that HER3T355I induced cell proliferation is via HER4/HER1-dependent ERK1/2 and cyclinD1 mediated pathways in ER+ cells. ERα expression is upregulated in ER+ cells expressing HER3T355I mutant. We noted crosstalk between ERα and HER3 in T47D cells. Several HER3 mutants (F94L, G284R, D297Y, T355I, and E1261A) acquired a gain-of-function phenotype in MCF10AHER2 cells and were resistant to lapatinib. These mutants increased HER2-HER3 heterodimerization. Knocking down HER3 from ovarian and colorectal cancers with endogenous HER3 mutations abrogated cancer cell proliferation. Overall, this study provides the first systematic assessment of how mutations in HER3 affect response of ER+ and HER2+ breast cancers to clinically relevant inhibitors and finds that HER3 mutations can be activating independent of HER2 over-expression.